Expression and purification of human interleukin-3 and muteins thereof

ABSTRACT

Methods are provided for improved production of hIL-3 either in glycosylated form from mammalian and yeast cells or in unglycosylated form from prokaryotes. Recombinantly produced human IL-3 is purified in a series of steps, initially employing hydrophobic interaction, followed by ion exchange chromatography and gel filtration.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 07/249,184, filed 16 Aug. 1988; now abandoned and PCTApplication No. NL87/00037, filed 16 Dec. 1987.

FIELD OF THE INVENTION

The present invention relates to cDNA encoding human interleukin-3(IL-3) and its use, inter alia, in cloning and expression in variousorganisms, including microorganisms, in particular yeasts, bacteria andfungi, tissue culture cells and transgenic animals and plants. Theinstant invention also relates to improved methods for the productionand purification of hIL-3 and muteins thereof.

BACKGROUND OF THE INVENTION

Hemopoiesis involves the active process of proliferation anddifferentiation of pluripotent progenitor cells into all types of matureblood cells and some specialized tissue cells. Production of functionalblood cells is regulated by specific proteins, the hemopoietic growthfactors (HGFs). Some of the HGFs control maturation of a specificmaturation lineage, whereas others stimulate proliferation anddifferentiation of progenitors along multiple pathways. Much of ourknowledge of the hemopoietic differentiation process has been obtainedfrom mouse studies in vitro and in vivo, using purified growth factors.The murine growth factor interleukin-3 (mIL-3), also termed multi-CSF,mast cell growth factor, stem cell activating factor or several otherdesignations, stimulates the proliferation of developmentally early,multipotent cells (CFU-S) as detected by the spleen colony assay,resulting in the production of progenitor cells along the erythroid,megakaryocyte, granulocyte/macrophage, osteoblast and several otherlineages. Furthermore, mIL-3 has been implicated in replication ofpluripotent stem cells, probably in synergism with other HGFs.

In recent years, several groups have succeeded in cloning mIL-3 cDNA. Noresults have been reported so far of identifying homologous sequences inhuman DNA using mIL-3 DNA as a probe. Presumably, the human gene hasdiverged extensively from the mIL-3 gene or has lost its function duringprimate evolution. However, human leukocytes were found to produce anHGF(s) which can replace M-GSF in supporting the proliferation of murineCFU-S. Thus, the existence of a human HGF was postulated, which sharesbiological properties with mIL-3 and therefore could be the humanhomolog.

Recently, DNA sequences encoding hIL-3 have been identified by severalinvestigators. For instance, using as probe a cDNA coding for gibbonIL-3, the human IL-3 gene was isolated (Yang et al., 1986). The sequenceof the exons of the human gene was disclosed in the cited paper as wellas in patent application WO 88/00598 (published 28 Jan. 1988). However,as known to those skilled in the art, the intron-containing genomicsequence cannot be used for synthesis of hIL-3 in microorganisms.Rather, the coding sequence used should be a continuous coding sequenceas in a cDNA. A cDNA sequence encoding human IL-3 is also disclosed inWO 88/00598. Following another route, Dorssers et al. (1987) alsoisolated a cDNA coding for human IL-3.

Patent Application No. WO 88/05469 discloses the isolation of a cDNAencoding hIL-3 using a synthetic DNA derived from the genomic sequencedescribed by Yang et al. (1986) as a probe. The disclosed cDNA sequence,however, lacks two amino acids, nos. 44 and 45 or 45 and 46. The aminoacid bordering either deletion is a GAC encoded Asp. Nonetheless, theculture supernatant of a yeast transformant carrying this cDNA sequencein an expression cassette, encoding mature hIL-3 fused to an N-terminal"flag" of 8 amino acids, shows IL-3 activity in a human bone marrowproliferation assay. This finding indicates that the absence of theaforementioned two amino acids and the N-terminal extension of 8 aminoacids has no deleterious effect on the biological activity of theprotein.

Finally, EP 282.185 also discloses the isolation of a hIL-3 cDNAsequence using as probe a synthetic DNA derived from the genomicsequence described by Yang et al. (1986) and describes the constructionof a completely synthetic hIL-3 coding sequence as well as theconstruction of two muteins, Ile² and Leu¹³¹. There is no mention ofbiological activity. Furthermore, it was apparently assumed that hIL-3contains 132 amino acids, starting at the N-terminus with Pro¹ -Met² -,whereas it is generally accepted that hIL-3 is 133 amino acids long andhas as the N-terminus Ala¹ -Pro² -Met³.

It is noteworthy that Yang et al. (1986) find a Ser residue at position8 of the mature hIL-3, whereas all other references indicate thepresence of a Pro at this position.

BPV-1 or the 69% subgenomic fragment (BamHI-HindIII) has been used forthe expression cloning of a variety of genes in different cloningsystems. EP-A-198386 describes the expression of gamma-interferon inC127 mouse cells. In EP-A-105141 the use of the BPV vector is describedfor the expression of hepatitis B surface antigen (HBsAg) in vertebratecell lines e.g. NIH 3T3, LTK⁻ mouse fibroblasts and African green monkeykidney cells. The general idea of using BPV-1 is disclosed in U.S. Pat.No. 4,419,446.

BPV-1 is one of at least six bovine papillomaviruses and is associatedwith cutaneous fibropapillomas in cattle. These viruses can readilytransform a variety of rodent cells in culture. The molecularly clonedbovine papillomavirus DNA as well as a cloned 69% subgenomic fragmentare efficient in inducing transformed foci. Transformed cells containmultiple copies (10 to 120 per cell) of the viral DNA as unintegratedmolecules (Law et al., 1981). The genetics of bovine papillomavirus typeI have been extensively studied (for a review see Lambert et al., 1981).The BPV-1 genome is a circular, 7946 base-pair, double-stranded DNAmolecule. The transcription is complicated because of the presence ofmultiple promoters, splice sites, and differential production of RNAspecies. The activities of some of the promoters are under tight controlof transcriptional enhancers.

The so-called E2 (=early) ORF is very important in this respect. Thefull-length E2 ORF encodes a transactivating protein (E2-ta) which canstimulate transcription of the early genes.

This protein consists of two conserved domains, the amino terminaldomain (which has transactivating activity) and the carboxy-terminaldomain (which has both DNA-binding and dimer formation activities). TheE2 ORF encodes a second regulatory protein, the E2 transcriptionalrepressor (E2-tr), which is an amino-terminally truncated form of theE2-ta protein. E2-tr is encoded by another mRNA, whereby the translationinitiation codon is an E2 ORF internal ATG-codon.

The present invention discloses cell lines not previously employed inhIL-3 production as well as mutations of the E2 ORF.

The clinical utility of hIL-3 is not only dependent on its inherentcharacteristics but also on its availability and the lack ofcontaminants. The prior art relating to the purification of murine,gibbon and human IL-3 is briefly reviewed here.

The mature murine T-cell enzyme marker 20a-hydroxy-steroid dehydrogenase(20aSDH) was found to be inducible in vitro. The factor responsible forthis was partially purified from splenic lymphocytes by Ihle et al.(1981). It was distinct from other known lymphokines in both itsbiochemical and functional properties. Ihle et al. (1981) proposed theterm interleukin-3 ("IL-3") for this factor. The purification bySephadex G-100 and DEAE cellulose chromatography resulted in a 9000-foldpurification, yet the final preparation still contained multipleproteins.

An improved purification procedure was presented by Ihle et al. (1982),wherein WEHI-3 cells which constitutively produce IL-3, were used. Here,through the extension of the earlier procedure with hydroxylappatite andreverse-phase high performance liquid chromatography, the final productcould be obtained 1,800,000-fold purified (their Table I). This productwas claimed to be homogeneous.

Miyajimi et al. (1987) used the silkworm Bombyx mori and an insectbaculovirus vector for high-level expression and secretion of murineIL-3. Purification of IL-3 from tissue culture medium was carried out bysequential passage through DEAE-Sephadex, ACA 54 and C8 reverse-phasecolumn chromatography. To obtain separation of three species of IL-3(18, 20 and 22 kDa) a second C8 reverse-phase column was necessary. Thedifferent species are due to differential glycosylation, sinceN-glycanase treatment yielded one final band of 15 kDa.

Ziltener et al. (1988) described the isolation of multiple glycosylatedforms of IL-3 by affinity purification. The observed microheterogeneitywas dependent on the source (activated T-cells, WEHI-3B cells or COS 7cells).

All of the above procedures describe the purification of murine IL-3. Inspite of the observed similarity of murine and human IL-3 with respectto their proliferative action on haematopoietic progenitor cells, thestructural homology between both proteins is rather low (28% at aminoacid level). This heterogeneity is illustrated by the total absence ofreactivity of the human protein on murine cells (and vice versa). Basedon their specific amino acid composition, the proteins likely requiredifferent methods of purification.

The purification of both gibbon and human IL-3, which show a structuralhomology of 93% (at the amino acid level), is disclosed in severalpatent publications. WO 88/00598 describes the isolation of a partiallysynthetic hIL-3 from the inclusion bodies of E. coli cells. The cellsare first disrupted by two passages through a french press, and theinclusion bodies are isolated by centrifugation in a sucrose stepgradient. This reference describes also three procedures for purifying ahuman or gibbon IL-3-like polypeptide from COS cell conditioned medium.In all cases a one-column process is used: either ion exchange or alentil lectin column or reversed-phase HPLC. The maximum purity obtainedfor gibbon IL-3 as determined with automated Edman degradation was 98%.

WO 88/05469 describes the purification of human IL-3 from yeast strainsby single or sequential reversed-phase HPLC steps. Since additional HPLCsteps can be employed if indicated, it is clearly not assumed that theproduct is homogeneous. No test of the purity of the hIL-3 wasdescribed, nor were data mentioned on the purity of the product.

Thus, no specific methods have been disclosed so far for thepurification of hIL-3. Therefore there is still a need for substantiallypure hIL-3 which can be used therapeutically and for a method ofpreparing such substantially pure product in a high yield, and which caneasily be scaled up.

SUMMARY OF THE INVENTION

The present invention describes the isolation of a cDNA comprising theentire coding sequence for human IL-3. The low degree of homologybetween the DNA sequences coding for murine and human IL-3 does notpermit the retrieval of a cDNA for hIL-3 by hybridization with the mIL-3coding sequence. Unexpectedly, the hIL-3 cDNA clone could be isolated byexploiting the rather high degree of homology in the 3' noncoding partof the cDNAs. The availability of the cDNA clone permits the productionof hIL-3 in a wide range of host organisms. Subsequent to large-scaleproduction, the protein may be purified and used therapeutically.

The present invention permits production of recombinant human IL-3protein in a wide range of host cells by transcription and translationfrom a cDNA sequence encoding the human IL-3 protein. The production ofthe protein is illustrated hereinbelow in several hosts, including E.coli, COS cells, CHO cells, C127 cells, FR3T3 cells, B. subtilis, B.licheniformis, S. cerevisiae and K. lactis. Production in other hostsusing appropriate expression systems is also made possible by provisionof the intronless cDNA. More generally, the availability of antihumanIL-3 antibodies which permit identification of colonies exhibitingsuccessful production of the recombinant protein aids in production ofhuman IL-3 from any recombinant system.

In one aspect, therefore, the invention is directed to a recombinant,intronless, DNA encoding human IL-3 protein.

In another aspect, it is directed to expression systems capable ofeffecting the expression of said DNA sequence encoding hIL-3 in anappropriate host.

In other aspects, the invention is directed to recombinant human IL-3protein in glycosylated or unglycosylated form, to purified human IL-3free of substances normally accompanying said protein, and to antibodiesspecifically reactive with these recombinant or purified proteins.

The invention also provides a method for purifying human IL-3 tohomogeneity by an initial stage of hydrophobic interaction, followed byion exchange chromatography and gel filtration. This method isparticularly useful for hIL-3 obtained by recombinant prokaryotic andeukaryotic expression systems. These and other aspects of the presentinvention will be further discussed below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B shows a comparison of DNA and protein sequences of humanmulti-CSF and mouse IL-3. The hmulti-CSF protein and DNA sequence (cloneD11, top lines) were aligned with the mIL-3 DNA (Fung et al., 1984;Miyatake et al., 1985) and protein sequence (Clark-Lewis et al., 1986).Identical nucleotides are indicated by a vertical line, identical aminoacids are shown in boxes. Black dots indicate a polyadenylation signalsequence and horizontal bars mark ATTTA repeat units.

FIG. 2 shows the construction of plasmid pLB4 containing human IL-3cDNA. E=EcoRI, Sm=SmaI, B=BamHI, S=SstI, K=KpnI.

FIG. 3 shows the biological activity of COS/pLB4 CM on human bone marrowprogenitors. The mean numbers of erythroid (BFU-E),granulocyte-macrophage (CFU-GM), granulocyte (CFU-G), eosinophil(CFU-Eo), macrophage (CFU-M) and mixed (CFU-MIX) colonies (±SD) areshown for duplicate cultures stimulated with graded volumes for COS/pLB4CM.

FIGS. 4A-4B shows induction of AML proliferation by COS/pLB4 CM asassessed in a colony culture assay (panel A) and in a DNA synthesis(3H-TdR incorporation) assay (panel B).

FIG. 5 shows a construction diagram of the E. coli expression vectorspGB/IL-301, pGB/IL-302, pGB/IL-303, pGB/IL-304, pGB/IL-305 andpGB/IL-306. In this figure, X stands for XhoI, E for EcoRI, B for BamHIand A for AvaI sites.

FIG. 6 shows the sequence of the multicloning site in pTZ18R (Pharmacia)and its derivative pT1.

FIG. 7 shows a schematic presentation of hmulti-CSF expression clones.For the eukaryote expression plasmids pLB4 and pLH1 only the multi-CSFcDNA insert is shown. Leader peptide ( ) and mature multi-CSF protein () coding regions are indicated in boxes. Bacterial expression clones ofhmulti-CSF (derived from pLH1) contain the lacZ and multi-linker proteincoding region ( ), the 5' terminal noncoding region of hmulti-CSF ( )and the hmulti-CSF coding region. The arrow marks the ATG start codonused in the particular vector.

FIGS. 8A-8B shows the sequences of fusion regions of lacZ/hmulti-CSF DNAfor various bacterial expression vectors. The sequence of clones isgiven from the start of the lacZ protein in either pUC8 or pTZ18R (lowercase letters) and of hmulti-CSF DNA sequence up to the ClaI site atposition 158. Mutations in the hmulti-CSF DNA sequence are underlined,resulting in: trp¹³ →arg¹³ (pGB/IL-302); leu⁹ →pro⁹ and trp¹³ →arg¹³(pGB/IL-303); met³ →thr³ and a silent change (pGB/IL-304). Thesuperscripts denote the amino acid residue number of the mature protein.

FIG. 9 shows polyacrylamide gel-electrophoresis of bacterial hmulti-CSFproduced from bacteria containing pGB/IL-301 and pGB/IL-302.

FIG. 10 shows the titration of hmulti-CSF fusion protein on AML blastcells.

FIG. 11 shows a Western blot demonstrating the IL-3 specific reaction ofrabbit antisera raised against the 21 kd protein isolated from a lysateof E. coli transformed with pGB/IL-301.

FIGS. 12A and 12B show the effect of the antisera of FIG. 11 on IL-3activity.

FIG. 13 shows a schematic representation of plasmid pGB/IL-307. The box( ) indicates the human Il-3 coding sequence. The N-terminal amino acidsof the fusion protein are depicted below the drawing.

FIG. 14 shows a schematic of plasmid pGB/IL-308. The nucleotide sequenceof the promoter region is depicted below the drawing.

FIG. 15 shows construction of plasmid pGB/IL-309. The first box ( )indicates a part of the human IL-3 sequence, viz., the signal sequenceplus 20 amino acids of the mature protein. The other box ( ) indicatespart of the 3' noncoding region of the IL-3 cDNA sequence.

FIG. 16 is a schematic representation of plasmid pGB/IL-310.

FIGS. 17A-17F shows the nucleotide sequence of plasmid pBHA1.

FIG. 18 shows the construction of the plasmids pGB/IL-311 andpGB/IL-312. The box ( ) indicates the precursor human IL-3 codingregion.

FIG. 19 shows the construction of the plasmid pGB/IL-313. The sequenceat the 5' side of the IL-3 sequence is depicted below the drawings.

FIG. 20 shows a schematic representation of plasmid pGB/IL-317construction.

FIG. 21 shows a schematic representation of plasmid pGB/IL-316construction.

FIGS. 22A-22B shows the nucleotide sequence of plasmid pGB/IL-316between the unique SacII site in the lactase promoter and the HindIIIsite behind the terminator (residues 4457 to 7204).

FIGS. 23A-23B shows the nucleotide sequence of plasmid pGB/IL-318between the unique SacII site in the lactase promoter and the HindIIIsite behind the terminator (residues 4457 to 7190).

FIG. 24 shows the nucleotide sequence of the EF-1alpha promoter,SalI-BglII-XhoI linker, and actin terminator as present on plasmidpGB/TEFact.

FIG. 25 shows the number of leukocytes and thrombocytes in chimpanzeeblood after subcutaneous injection of 30 ug/kg IL-3, during day 0 to 6.

FIG. 26 shows the elution profile of the hydrophobic interactionchromatography of hIL-3 from Bacillus licheniformis T9 on FractogelTSK-butyl 650C (d×h 25×8 cm). The hIL-3 containing fractions areindicated with a horizontal bar.

FIG. 27 shows the elution profile of the anion exchange chromatography(first run) of hIL-3 from Bacillus licheniformis T9 on Q-Sepharose FastFlow (d×h 10×11 cm).

FIG. 28 shows the elution profile of the anion exchange chromatography(second run) of hIL-3 from Bacillus licheniformis T9 on Q-Sepharose FastFlow (d×h 5×90 cm).

FIG. 29 shows the elution profile of the gel filtration chromatographyof hIL-3 from Bacillus licheniformis T9 on Sephacryl S100 HR (d×h 5×90cm).

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

As used herein, "human IL-3", "hIL-3", "human multi-CSF", and"hmulti-CSF" are used interchangeably, and designate a proteinpreparation which exhibits the following activities:

1. The protein stimulates colony formation by human hemopoieticprogenitor cells wherein the colonies formed include erythroids,granulocytes, granulocyte macrophages, megakaryocytes and mixturesthereof.

2. The protein stimulates DNA synthesis by human acute myelogenousleukemia (AML) blasts, as evidenced, for example, by labeled thymidineuptake.

To fit the definition of hmulti-CSF, the activity in the foregoing assaymust not be substantially inhibited by antibodies raised in response to,and immunospecific for, GM-CSF, unless these antibodies also inhibitthese activities by the illustrative hmulti-CSF below.

By a protein "equivalent to hIL-3" is meant a protein displaying theabove biological activity.

One illustrative form of hmulti-CSF is shown in FIG. 1 as a 133 aminoacid mature protein, having a 19 amino acid signal sequence. The aminoacid sequence of FIG. 1 is identical with that disclosed by Yang et al.(1986) except at position 8 of the mature protein wherein the Ser of theYang protein is replaced by Pro herein. As shown herein, this amino acidsequence is effective in its nonglycosylated form. However, it containstwo glycosylation sites, and the glycosylated form is also includedwithin the scope of the invention. It is also recognized that theprotein may exist in acid addition salt form, basic salt form, or may beneutral, depending upon the pH of its surroundings. Derivatization byphosphorylation, acetylation, and so forth to the extent that activityis not destroyed, also results in a protein included within the scope ofthe invention.

It is also recognized that the entire sequence may not be necessary foractivity. Parts of the amino acid sequence may be deleted or replaced,while retaining biological activity. As illustrated herein, the alanineat position 1 may be deleted, as may as many as the first fourteen aminoacid residues if replaced by a sequence of residues of a fused peptidesequence. In addition, it is believed that the murine form of theprotein requires only the first 79 residues for activity; thiscorresponds approximately to the first 83 residues of the humancounterpart. Accordingly, fragments which comprise only the first 83amino acid residues of the protein, and the N-terminal replaced formsthereof, are also included within the scope of the invention.Furthermore, it should be considered that the N-terminus of mature hIL-3is formed by the residues ala-pro-met etc. (see FIG. 1). It is knownthat the protein, when secreted by a yeast host, may in some instancesbe shortened by two amino acids (ala-pro), due to the interaction with adipeptidylaminopeptidase (Suarez Rendueles, 1981). The hIL-3 without theN-terminal alanine and proline still retains its biological activity.Yeast strains carrying a null mutation of the X-prolyldipeptidylaminopeptidase gene will produce complete hIL-3 (amino acids1-133). Accordingly, included in the multi-CSFs of the invention arethose which contain and those which do not contain the N-terminalalanine and proline, produced by X-prolyl dipeptidylaminopeptidasemutants and wild-type hosts, respectively.

When produced as a mature protein in a prokaryotic host, the codingsequence for the mature protein will be prefaced by an ATG start codon.The resulting N-terminal methionine may then be removed, or partiallyremoved, by processing within the bacterial host, depending on thenature of the subsequent amino acid sequence. Again, both forms of thehIL-3 are biologically active. Therefore, included in the hmulti-CSFs ofthe invention are those which contain and those which do not contain theN-terminal methionine.

From the above it is clear that amino acid changes may be introducedinto the human IL-3 protein without affecting its biological function.It is recognized that minor changes in amino acid sequences by chemicalmodification of the encoded residue, substitution of a differentresidue, or deletion or addition of one or more, but preferably onlyone, residue results in proteins which retain activity. Accordingly,these nondestructive mutations are also included within the invention,in particular, the naturally occurring allelic variations and othermutations which are nonlethal to the activity.

On the other hand, it should be considered that amino acid changes inthe human IL-3 protein may be beneficial to the therapeutic use of theprotein. As recognized herein, the mature protein has four conserveddomains at residues 15-36, 54-61, 74-91, and 107-118. Proteinscontaining single and multiple amino acid changes in the nonconservedregions, 1-14 (which are, in any event, replaceable by the sequences ofhost-derived fusion proteins), 37-53, 62-73, 92-106, and 119-133, arepossible. However, it appears that the cysteine residues at positions 16and 84 may be necessary for disulfide bridge formation as they areconserved between species. Changes in the conserved domains mentionedabove may influence biological properties of the protein, such asreceptor binding and signal transduction. It is envisaged that hIL-3having altered properties are of therapeutic use. Such derivatives ofhIL-3, which may be made by known protein engineering techniques, are tobe understood to be within the scope of the present invention.

hIL-3-like proteins, with altered amino acid sequences, may have alteredeffects on target cells. Such molecules may function either as agonistsor as antagonists and have potential clinical applications. Thesealtered molecules are included in the present invention, and several ofthese hIL-3-like proteins are biologically active. These active proteinsmay serve as agonists and may show properties beneficial in clinicaluse, such as enhanced stability, better binding to the IL-3 receptor,etc. Proteins which have lost their biological functions may still serveas antagonists, e.g., when binding of such a protein to the IL-3receptor does not result in signal transduction. These proteins are alsocontemplated by the present invention.

The protein preparation may contain the hmulti-CSF peptides in monomericor aggregated form, provided the aggregates retain activity as definedabove.

As used herein, "expression system" refers to a DNA sequence whichcontains both a coding sequence whose expression is desired andappropriate control sequences in operable linkage with it which permitsits expression when the control sequences are compatible with the hostinto which the expression system is placed. As is generally understood,"control sequences" refers to DNA segments which are required for orregulate the expression of the coding sequence with which they areoperably linked.

Control sequences for all hosts include promoters, which may or may notbe controllable by regulation of their environment. Typical promoterssuitable for prokaryotes include, for example, the trp promoter(inducible by tryptophan deprivation), the lac promoter (inducible withthe galactose analog IPTG), the beta-lactamase promoter, and thephage-derived P_(L) promoter (inducible by temperature variation).Additionally, especially for expression in Bacillus, useful promotersinclude those for alpha-amylase, protease, Spo2 and synthetic promotersequences. Suitable promoters for expression in yeast include the3-phosphoglycerate kinase promoter and those for other glycolyticenzymes, as well as promoter regions for alcohol dehydrogenase, andyeast phosphatase. Also useful are the transcription elongation factor(TEF) and lactase promoters. Mammalian expression generally employspromoters derived from viruses such as the adenovirus promoters and theSV40 promoter systems, but they also include regulable promoters such asthe metallothionein promoter, which is controlled by heavy metals orglucocorticoid concentration. There are also now available virus-basedinsect cell expression systems, as well as expression systems based onplant cell promoters such as the nopaline synthetase promoters.

In addition to the promoter DNA sequence, which is necessary for thetranscription of the gene by RNA polymerase, a variety of controlsequences, including those regulating termination (for example,resulting in polyadenylation in eukaryotic systems) are also useful incontrolling expression. Some systems also contain enhancer elementswhich are desirable but not necessary in effecting expression.

Translation controls include a ribosome binding site (RBS) inprokaryotic systems, whereas in eukaryotic systems translation may becontrolled by the nucleotide sequence around the AUG codon.

As implied above, recombinant protein production can be effected in awide variety of hosts, including bacteria (predominantly E. coli,Bacillus, and Streptomyces), in yeast and fungi (such as Saccharomyces,Kluyveromyces, and Aspergillus), and in mammalian and other cellcultures such as COS cells, C127 cells, FR3T3 cells, Chinese hamsterovary cells, Spodoptera frugiperda (Sf9) cells, and so forth. Theprotein may be produced as an intracellular mature or fusion protein, ormay be secreted when the DNA encoding an appropriate compatible signalis included in the gene.

It has been surprisingly found that the combination of a perfect fusionof host signal sequences to mature hIL-3 coding sequences, inconjunction with strong promoters in Bacillus, as well as the use ofstrong promoter/enhancer combinations and mRNA stabilizing sequences onBPV-1 derived vectors in C127, FR3T3 and CHO cells, leads to improvedproductivity of hIL-3.

All elements used for expression of hIL-3 in Bacillus species have beendescribed in WO 88/04691. Surprisingly, however, optimal combinations ofpromoters, signal sequences and mature hIL-3 coding sequences were foundby rearrangement of the different genetic elements. For proper secretionof hIL-3 by Bacillus species a perfect junction between a-amylase signalsequence and hIL-3 coding sequence was found to be crucial. Alreadyusing the sigma-43 promoter (see WO 88/04691) hIL-3 could be found inthe culture medium, but only after inclusion of the strong a-amylase orHpaII promoter in the expression plasmid high level expression wasobtained.

WO 88/04691 also describes expression of active hIL-3 by C127 cells.However, expression levels are relatively low when using the pLB4/BPVvector. It has now been surprisingly found that the use of thecombination of The Moloney Murine Sarcoma Virus (MSV) enhancer and mousemetallothionein I promoter (MT promoter), or the Human Cytomegalovirusenhancer/promoter, instead of the SV40 enhancer/promoter as present inpLB4/BPV, results in improved expression levels of hIL-3 by mammaliancells. Specifically, when an expression vector containing the MSVenhancer/MT promoter is used to establish stable cell lines from C127cells, a more than 20-fold increase in IL-3 expression can be obtained.These latter cell lines allow efficient production of hIL-3 withmammalian (complex) type glycosylation.

Furthermore, production of hIL-3 using stable CHO cell lines, describedherein, unexpectedly equalled the best known production system. TheseCHO cells are also readily adapted to growth in suspension and can bemass-cultured in large reactors.

Additionally, replacement of the hIL-3 cDNA sequence by the genomichIL-3 sequence has a beneficial effect on the steady state level ofhIL-3 mRNA.

A further increase in mRNA level can be achieved by mutating the startcodon used for the translation of E2-tr. From the description of BPV-1,it is already clear that E2-ta and E2-tr have an antagonistic activity.By mutating the aforementioned start codon, the production of E2-tr (therepressor protein) is clearly abolished. Without this repressoractivity, the transcription of the early genes is considerably increasedleading to an increase in plasmid copy number. This results in anincrease of the expression of the cloned gene and thus in a higherprotein yield.

The present invention, thus for the first time, enables large-scaleproduction of recombinant human IL-3, so that this protein--in purifiedform--can now be used as a therapeutic agent. The methods describedherein provide means for producing glycosylated as well asunglycosylated forms of the protein, which can be purified tosubstantially pure human IL-3. "Purified" human IL-3 refers to humanIL-3 as defined above which is substantially free of other proteinswhich normally accompany it. That is, hIL-3 is substantially free ofother proteins when at least about 75% by weight of the total hIL-3 plusother proteins is hIL-3. Preferably, hIL-3 comprises at least 90% byweight of the protein in the composition. By "crude" protein product ismeant a protein product that is not comprised of substantially purehIL-3.

Human IL-3 can be purified to homogeneity using a combination ofhydrophobic interaction, ion exchange chromatography and gel filtration.Although many purification techniques have been known for quite sometime, this specific combination surprisingly proved to be extremelypowerful for obtaining substantially pure hIL-3. Moreover, thispurification method results in a pyrogen-free product. It is verysuitable for upscaling after fermentation where hIL-3 contained in thefermentation broth after secretion from the cells must be purified fromcontaminating components of the growth medium. The method isparticularly useful for the purification of hIL-3 produced bytransformed hosts, both prokaryotic and eukaryotic.

The initial stage of the purification is preferably carried out byhydrophobic interaction. This step is unusual but very effective for thepurpose of this invention. Presumably, it causes a selective separationon the basis of the structure of the protein itself. The next step ispreferably ion exchange chromatography, in which hIL-3 is found in therun-through fractions separated from most of the contaminating proteins.Subsequently, gel filtration is applied which will separate lowmolecular proteins.

If hIL-3 degradation products are present in the starting material to bepurified, for example from Bacillus licheniformis, these proteins can beadvantageously separated from native hIL-3 by means of anion exchangechromatography, preferably on Q Sepharose Fast-Flow. Chromatography onhydroxylapetite and chromatofocussing on pBE94 are efficient techniquesfor the separation of hIL-3 degradation products.

The hydrophobic interaction is advantageously performed using aTSK-butyl or octyl Sepharose column, of which hIL-3 can be eluted forexample with gradients of either (NH₄)₂ SO₄ in Tris-HCl or ethyleneglycol. The removal of contaminating proteins (and other substances) byion-exchange chromatography may require the use of columns such as, forexample, TSK-DEAE, Q Sepharose Fast-Flow or TSK-CM.

Finally, gel filtration using, preferably, Biogel A or Sephacryl S100 HRis an excellent final step in the purification of unglycosylated hIL-3.

The starting point for the purification can be either intracellularhIL-3 or hIL-3 contained in a fermentation broth. The isolation ofintracellular hIL-3 is made much easier when the protein is contained inso-called inclusion bodies. The first step would then be the isolationof these bodies which contain the product in a relatively pure form at ahigh concentration. After solubilization, hIL-3 can be further purifiedby chromatography.

B. Retrieval of cDNA Encoding Human IL-3

Human IL-3 cDNA was isolated according to the following strategy:

1. A procedure was developed which allowed for reproducible productionof hemopoietic growth factors (HGFs) by human leukocytes.

2. mRNA was prepared from such producing cells and transcribed intodouble-stranded cDNA.

3. The cDNA was screened with a complete mIL-3 cDNA which contained boththe coding and untranslated 3' downstream portions to obtain DII.

4. The hybridizing cDNA clone D11 was inserted into an expression vectorpLO to obtain pLB4 which was expressed in COS cells to confirm thepresence of the sequence encoding human IL-3. Conditioned media fromthese cells showed the biological activity expected of hIL-3.

The human cDNA was retrievable using this procedure because despiteconsiderable lack of homology with the murine coding sequence, asurprising degree of homology was present in the 3, untranslated region.Applicants are unaware of any prior disclosure of the use of a 3'untranslated sequence homology to retrieve an alternate species gene.

In more detail, conditioned medium of lymphocytes cultured in thepresence of 12-O-tetradecanoylphorbol-13 acetate (TPA) and concanavalinA (ConA) is a suitable source for human HGFs as determined by assay ofthe medium using stimulation of mouse CFU-S in suspension cultures,proliferation of mIL-3 dependent DA-1 cells, human hemopoieticprogenitor assays by colony formation in vitro, and in vitro stimulationof acute leukemia blasts. A cDNA library from human lymphocytes wasconstructed in lambda gt10 phage (Huynh et al., 1985) and screened usingthe HindIII-XbaI fragment of mIL-3 cDNA, for the occurrence of mIL-3related sequences. No hybridizing clones were identified.

However, when complete murine IL-3 cDNA was used as probe, four cloneswere identified. Restriction enzyme analysis of the largest clone (D11)indicated a 910 bp insert containing an internal EcoRI site (at position411, FIG. 1).

(It was investigated whether this EcoRI site had arisen by ligation oftwo independent cDNA fragments or was a naturally occurring site.Southern analysis of restriction enzyme digested human DNA using labeled5' and 3' EcoRI fragments of clone D11 as probe, revealed identical DNAfragments following digestion with HindIII (15 kb) and BamHI (4.6 kb).Furthermore, the DNA sequence around the EcoRI site does not correspondto linker sequence (pCCGAATTCGG) used for inserting cDNA into phage DNA,indicating that these EcoRI fragments are derived from a single mRNA.)

From hybridization and sequencing experiments it was concluded that thesmall clones (II, IV and VI) are identical to the 3' nucleotide sequenceof clone D11 and derived from the same mRNA species.

Computer assisted alignment (FIG. 1) of the D11 cDNA and the mIL-3 cDNArevealed sequence homology in the 5' terminal 100 bp, betweennucleotides 236-269 and between nucleotides 598-803 in the 3' terminalregion (68%, 71% and 73% homology, respectively). In particular, theregion between the nucleotides 706 and 763 is highly conserved (93%homology) and contains repetitive AT-rich sequences. The low homology inthe 5' terminal 600 bp of the human cDNA (52%) precludes detection byhybridization with the HindIII-XbaI fragment of mIL-3.

Analysis of the human cDNA clone for an encoded protein shows an openreading frame up to the termination codon TGA at position 495-497 (FIG.1). The first ATG triplet is probably the actual initiation codon of theencoded polypeptide. The putative encoded protein consists of ahydrophobic leader peptide of 19 amino acids, which is probably cleavedbetween the glycine and alanine residues (Von Heijne, 1983; Perlman andHalvorson, 1983).

The alignment of the predicted amino acid residues of the human andmouse IL-3 (FIG. 1) reveals a homology of 50% for the leader peptide(residues -19 to +1) and 28% for the mature protein (residues 1 to 133).Within the leader peptide, there are two conserved regions of four aminoacids (residues -13 to -10 and -3 to +1), of which the second oneencloses the processing site. The mature protein is 133 amino acids longand has a molecular weight of 15 kd. The mature protein has fourconserved domains (residues 15-36, 54-61, 74-91 and 107-118) andcontains two potential glycosylation sites (residues 15-17 and 70-72).Both cysteine residues present in the human protein (positions 16 and84) are conserved and may play an essential role in protein folding bydisulfide bridge formation.

In order to verify that this human cDNA encodes a functional proteinthat resembles mIL-3, the D11 cDNA was inserted in a eukaryoticexpression vector (pLO containing an SV40 transcription unit) to obtainthe expression vector pLB4 and transfected to COS 1 cells. The COS/pLB4conditioned medium (CM) was tested for (1) its capacity to stimulatecolony formation by human bone marrow cells, and (2) to stimulate humanacute myelogenous leukemia (AML) blasts.

In vitro colony growth of human hemopoietic progenitors depleted ofmyelomonocytic (Vim-2 positive) and T-lymphocytic (T-3 positive)accessory cells, was efficiently stimulated by COS/pLB4 CM. The datademonstrate stimulation of progenitors of several hemopoieticdifferentiation lineages and of a subpopulation of BFU-E by COS/pLB4 CM.

In a separate experiment, bone marrow was enriched for progenitor cellsby density centrifugation, E-rosette sedimentation to removeT-lymphocytes and adherence to remove mononuclear phagocytes and culturein enriched medium containing fetal calf serum. Under these conditions,the majority of the colonies obtained upon stimulation with COS/pLB4 CMcontained two or more hemopoietic differentiation lineages: allcontained macrophages, approximately half immature blasts and/orimmature erythroid cells and/or neutrophilic granulocytes and aminority, in addition, basophilic or eosinophilic granulocytes. Theseresults demonstrate the multilineage stimulatory properties of theprotein encoded by the human cDNA clone D11 and its action ondevelopmentally early, multipotent hemopoietic cells.

With respect to AML stimulation, AML blasts of five patients werestimulated with the COS/pLB4 CM and assayed for a response by measuring³ H-TdR incorporation and colony formation. Three of the five leukemiacell samples responded to the COS/pLB4 CM in both assays; characteristicdose-response relationships for colony formation and DNA synthesis ofAML blasts of different patients were obtained. The responses to GM-CSFdemonstrated further phenotypic differences among the leukemiasresponding to the COS/pLB4 CM.

These data demonstrate that the D11 cDNA clone contains the completegenetic information for a biologically active protein which is exportedinto the culture medium in the transformed COS cells. Despite theapparent lack of homology with respect to the protein sequence betweenthe human protein and mIL-3 (less than 30%), the proteins are comparablewith respect to their biological function. Both proteins exert theireffect on developmentally early hemopoietic progenitors of variouslineages. The low homology at the amino acid level is also reflected ina low homology in the coding nucleotide sequence. However, veryunexpectedly, a rather high degree of homology--sufficient for retrievalof the human cDNA clone--occurred in the 3' untranslated region.

Southern analysis of human DNA revealed a single hybridizing geneindicating that this cDNA does not belong to a family of closely relatedgenes.

From the foregoing results we conclude that the human cDNA insert in D11encodes the human homolog of mIL-3. We decided to use the operationalterm hmulti-CSF for the protein encoded by the cDNA clone D11 in view ofits major biological effect and assay.

The identification of hmulti-CSF cDNA clones by virtue of hybridizationwith the 3' terminal region of the mIL-3 cDNA was unexpected. Whereashomologous DNA sequences are in general predominantly in the codingregion, the hmulti-CSF sequence has extensively diverged (45% homology)in this part of the gene. Analysis of the highly conserved domain in the3' terminal noncoding region reveals the occurrence of 5 ATTTA repeatunits which are all preserved in the mIL-3 cDNA (FIG. 1).

hmulti-CSF and mIL-3 display considerably less protein homology thanother murine and human growth factors or lymphokines such as GM-CSF(Schrader et al., 1986), interleukin-2 (Schrader et al., 1986),interleukin-1 (March et al., 1985) and interferons (Higashi et al.,1983; Dijkema et al., 1985; Zwarthoff et al., 1985). The biologicalactivity of the mature mIL-3 appears to be contained in the first 79amino acids, including an absolute requirement for the cysteine residueat position 17 (Clark-Lewis et al., 1986). This cysteine residue isconserved in hmulti-CSF (FIG. 1, pos. 16) and may play an essential rolein protein folding. The occurrence of a potential glycosylation sitearound this cysteine residue may interfere with disulfide bridgeformation.

C. Production and Formulation of hmulti-CSF

Applicants have provided a representative variety of expression systemscapable of producing human IL-3 protein in a variety of forms--as fusionproteins, as mature intracellular proteins, and as secreted proteins.Applicants are unaware of availability anywhere in the art ofrecombinant forms of human IL-3, or, indeed, of any human IL-3 in apreparation which is free of proteins normally accompanying this desiredprotein. Accordingly, the invention herein provides, for the first time,the human IL-3 protein in a manner which is capable of adaptation totherapeutic and diagnostic uses.

The human IL-3 can be produced as a fusion protein with sequencesheterologous to the human IL-3 amino acid sequence. By "heterologous" ismeant a sequence which is not found in human IL-3 itself, but is anunrelated sequence. This heterologous sequence may be derived from abacterial protein, a yeast protein, a mammmalian protein, or any of avariety of miscellaneous fortuitously encoded sequences such as, forexample, those encoded by polylinkers. It is clear from the resultshereinbelow that at least the first 14 amino acids of the N-terminus ofthe human IL-3 sequence can be replaced by a heterologous sequence, atleast if the fusion protein is further extended past the N-terminus.

Heterologous gene expression has been found to be a particularly usefulmethod for producing the subject proteins. For example, host signalsequences can be fused to mature hIL-3 coding sequences in conjunctionwith strong promoters in Bacillus. Likewise, strong promoter/enhancercombinations and mRNA stabilizing sequences on BPV-1-derived vectors insuch cell lines as C127, CHO and FR3T3 are also useful. These and otherheterologous expression systems are described more fully in theexamples.

The protein can also be obtained as a mature intracellular protein byconstructs in which the ATG start codon is placed immediately upstreamof the desired N-terminus. These intracellular proteins, whether matureor fusion proteins, can be recovered by lysing the cells and purifyingthe human IL-3 using standard protein purification techniques.

Protein purification is simplified if the human IL-3 is secreted intothe medium. When produced in mammalian cells with which the nativesignal sequence is compatible, this native signal sequence can be usedto effect secretion into the medium. In bacterial or yeast systems,signal sequences compatible with these hosts, such as the penicillinaseor alpha-amylase sequence in bacteria or the alpha-factor signalsequence in yeast can be used.

When produced recombinantly, the human IL-3 is free of proteins normallyaccompanying it, and can be purified from the proteins and othermaterials indigenous to the recombinant host using, for example,chromatographic methods, gel filtration, ammonium sulfate precipitation,and so forth. A combination of hydrophobic interaction, ion-exchangechromatography, and gel filtration has proved to be an extremelypowerful method for obtaining substantially pure hIL-3. The hydrophobicinteraction may be performed using a TSK-butyl or octyl Sepharosecolumn, on which hIL-3 can be eluted with gradients of either (NH₄)₂ SO₄in Tris-HCl or ethylene glycol. Contaminating substances can be removedby ion-exchange chromatography, using, for example, TSK-DEAE or TSK-CMcolumns. Finally, gel filtration using Biogel A has proved to be anexcellent step in purification of unglycosylated hIL-3.

As described hereinbelow, the protein is useful for therapeutic anddiagnostic purposes. For therapeutic uses, the protein may be formulatedin ways standard for pharmaceutical compositions which are used for theadministration of proteins. Suitable excipients include, for example,physiological saline, Ringer's solution, and so forth. Alternateformulations, including solid formulations (e.g., lyophilized), can alsobe employed.

D. Preparation of Antibodies

The availability of recombinant IL-3 protein or parts thereof willpermit production of antibodies directed against the protein or partsthereof, as demonstrated hereinbelow. Such antibodies are useful, interalia, for in vitro detection of colonies producing hIl-3, such as inquantitative or qualitative ELISA tests, for therapeutical use and forthe purification of both natural and recombinant hIL-3.

Statement of Utility

The nucleotide sequence of the whole or parts of the cDNA of human IL-3,or closely-related DNA sequences will advantageously enable thedetection of genetic abnormalities, including genomic rearrangements,restriction fragment-length polymorphisms, mutations and altered geneexpression with the use of such techniques as the analysis ofchromosomal DNA using restriction enzymes, DNA and RNA blotting as wellas hybridization techniques (Maniatis et al., 1982) and two-dimensionalgel electrophoresis (Fisher and Lerman, 1983).

The recombinant hmulti-CSF as provided by the present invention willfacilitate a detailed analysis of its role in human hemopoiesis, inparticular the possible synergism of hmulti-CSF and various other HGFs.Furthermore, hmulti-CSF is of considerable interest because of itsapplicability for in vitro diagnosis of human diseases in whichhemopoietic progenitor cells are involved, which include leukemia, aswell as potential therapeutic applications aimed at expansion ofhemopoiesis in vivo. The effect of hmulti-CSF on various hemopoieticmalignancies with respect to terminal differentiation of the leukemiccells also needs to be explored. In addition hmulti-CSF may be requiredfor establishing a proliferative state of human stem cells in genetherapy protocols, since stimulation with mIL-3 was shown to be requiredfor successful infection of mouse stem cells with recombinant,replication defective retroviruses.

IL-3 protein can also advantageously be used for the detection of earlyhemopoietic precursor cells in standardized in vitro cultures (Wagemakerand Visser, 1980; Merchav and Wagemaker, 1984; and Metcalf, 1986).

IL-3 protein and variants can further be used for the multiplication ofhemopoietic stem cells in vitro, possibly in conjunction with othergrowth factors, for bone marrow transplantation and the geneticmanipulation of stem cells (Lowenberg and Dicke, 1977; Wagemaker andPeters, 1978; Lemischka et al., 1986).

The IL-3 protein can be used for the determination of the responsepattern of malignant hemopoietic cells in in vitro tests (Touw andLowenberg, 1985; Griffin et al., 1986; Griffin and Lowenberg, 1986).

The IL-3 protein can further be used for the detection of remainingleukemic cells by in vitro methods (Touw et al., 1986; Griffin et al.,1986; Griffin and Lowenberg, 1986).

Furthermore, the IL-3 protein can be used in vivo for the treatment andprevention of malignant and nonmalignant disorders, either by itself orin combination, in which an obtained specific response by thehemopoietic system can result in a clinical benefit.

These applications include: cytopenias and/or immunosuppression due toinfections such as AIDS; cytopenias due to chemotherapy and/orirradiation; bone disorders such as bone fractures and osteoporosis;immunodeficiencies due to general anaesthetic procedures; recoveryfollowing bone marrow transplantation; adjunct to vaccinations andadjunctive therapy of infections.

The cloned human IL-3 DNA sequence or closely related DNA can be usedfor gene therapy in genetic deviations from the normal IL-3 gene.

To facilitate the above-described analysis, a large quantity of humanIL-3 is required. The easiest way to obtain sufficient amounts of theprotein is the production with microorganisms, in particular yeasts,bacteria and fungi, e.g., Saccharomyces, Kluyveromyces, Aspergillus,Streptomyces, Bacillus and E. coli species. Production in mammalian andother eukaryotic systems, such as C127 cells, CHO cells, FR3T3 cells,Spodoptera cells, and transgenic animals and plants, is also possiblefor skilled persons following the teaching of the present invention.These possibilities are all included within the scope of this invention.

As an illustration of how to obtain living cells that produce the humanIL-3 protein by expression of the hIL-3 cDNA, a number of plasmids wereconstructed and transferred to E. coli, B. subtilis, B. licheniformis,S. cerevisiae, K. lactis, C127 cells, CHO cells, and FR3T3 cells. Usingthese host strains, the production of recombinant human IL-3 wasachieved. The products were tested for their capacity to stimulate humanAML blasts as described above for the COS/pLB4-conditioned medium. Fromthese experiments it appeared that the proteins made were biologicallyactive.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 Retrieval of cDNA Encoding Human multi-CSF (hmulti-CSF)

Human leukocytes stimulated with TPA (5 ng/ml) and ConA (10 ug/ml)produced considerable amounts of HGFs as measured by the murine stemcell proliferation assay and various other colony assays. Cells wereharvested 24 hours after stimulation, because mRNA production is oftentransient following stimulation with phorbol esters and lectins. Alreadyafter 24 hours, HGFs were easily detectable in the CM.

mRNA Preparation

Cells were harvested, washed with PBS and homogenized in guanidiniumisothiocyanate solution (Maniatis et al., 1982). RNA was pelletedthrough a cesium chloride cushion. Oligo(dT)-cellulose chromatographywas used for selection of mRNAs (Maniatis et al., 1982).

cDNA Synthesis

cDNA was synthesized essentially according to Gubler and Hoffman (1983),using oligo(dT) as primer and AMV reverse transcriptase. Second strandwas synthesized with RNaseH and E. coli DNA polymerase I. Gaps wereclosed with T4-DNA ligase and ends were flushed by T4-DNA polymerase. Toprotect internal EcoRI restriction sites, the cDNA was methylated withEcoRI methylase. Subsequently, the cDNA was ligated to phosphorylatedEcoRI linkers with T4-DNA ligase. After digestion with EcoRI, excesslinkers were removed by Sepharose CL-4B chromatography. The materialrecovered in the void volume of the column was larger than 250 bp andwas used for construction of the libraries.

Construction of the Phage cDNA Library

The cDNA was ligated to lambda gt10 phage arms (Huynh et al., 1985) andpackaged with commercial packaging extracts (Gigapack, Vector CloningSystems). The recombinant phages were propagated in E. coli C600 hfl. clScreening of the Phage Library

Of each plate containing 1-5000 plaques, two nitrocellulose filterreplicas were made according to standard procedures. Filters were thenhybridized with radiolabeled mIL-3 probe from the HindIII-XbaI fragmentof mIL-3 cDNA or with the complete mIL-3 cDNA clone radiolabeled withrandom primers. The mIL-3 cDNA clone (pL101) was isolated from a WEHI-3BcDNA library. WEHI-3B mRNA was isolated using the guanidiniumisothiocyanate CsCl method, size fractionated on sucrose gradient andinjected into Xenopus laevis oocytes. RNA fractions inducing the oocytesto produce a factor capable of supporting murine stem cellproliferation, were used for synthesis of cDNA as described above, cDNAwas tailed with dC residues and inserted in the PstI site of pUC9. mIL-3clones were identified using synthetic oligonucleotides (from publishedmIL-3 sequence; Fung et al., 1984). Insert of pL101 was purified onpolyacrylamide gel and used for screening of the human cDNA library.Probe DNA was labeled using the random primer method (Feinberg andVogelstein, 1983). Potential positive plaques were rescreened and plaquepurified. In this way four clones were identified, including phage D11.

Sequencing of cDNA Clones

Recombinant phages were grown at large scale and purified, cDNA insertswere removed from the phage arms by digestion with EcoRI and purified onpolyacrylamide gel. The purified fragments were ligated into M13mp18 andpTZ18R DNA digested with EcoRI and used for transformation of E. coliJM109. Single-strand DNA was prepared and sequenced according toestablished procedures (Sanger et al., 1977). Sequence data wereanalyzed using various computer programs (Queen and Korn, 1984; Staden,1982; Devereux et al., 1984; Lipman and Pearson, 1985).

The sequence obtained for the insert in phage D11 is shown in FIG. 1.This 910 bp sequence contains the entire coding region for hmulti-CSFand its signal sequence, and exhibits high homology to the murine clonepL101 in the 3' untranslated region. The homology upstream in the codingsequence is relatively more limited. As described above, the protein hasa putative 19 amino acid signal sequence followed by a 133 amino acidmature protein containing two glycosylation sites (15-17 and 70-72) andtwo cysteine residues at 16 and 84.

The deduced amino acid sequence is the same as that encoded by thegenomic DNA disclosed by Yang et al. (1986) except for one aminoacid--that at position 8 of the putative mature protein; the Yang DNAencodes Ser, the cDNA herein encodes Pro.

The intronless sequence obtained in the phage D11 can be used forprokaryotic expression, as well as for expression in eukaryotic systems,as illustrated below.

EXAMPLE 2 Expression in Mammalian Cells A. Construction of The EukaryoteExpression Vector pLB4

Phage D11 (containing the longest cDNA insert) was digested with HindIIIand BglII and subcloned in plasmid pT1 (a derivative of pTZ18R,containing some additional restriction sites in the multilinker; seeExample 3A). Clones containing the phage fragment containing the cDNAinsert were identified by restriction analysis. The cDNA insert wasremoved from this plasmid by partial digestion with EcoRI and purifiedby polyacrylamide gel electrophoresis. The appropriate fragment wasinserted in a eukaryote expression vector (pLO) in an SV40 transcriptionunit.

pLO comprises: EcoRI (filled in)-PstI of pBR322 (1-755), PstI-AvaI ofpBR329 (756-1849), AvaI-PvuII adapter (1850-1868), PvuII-HindIII (filledin) of SV40 promoter (1869-2211), PvuII-BamHI adapter containing theunique EcoRI site (2211-2251), MboI "splice fragment" of SV40(2252-2861), BclI-BamHI (filled in) "poly A fragment" of SV40(2862-3098), PvuII-HindIII promoter fragment of SV40 (3099-3440),HindIII-BamHI Eco gpt gene (3441-4501), MboI "splice fragment" of SV40(4502-5111), and the BclI, BamHI (filled in) "poly A fragment" of SV40(5112-5348).

The Eco gpt transcription unit is of no importance in transientexpression of proteins in COS 1 cells. The resultant expression plasmidfor hmulti-CSF was termed pLB4 and was purified on CsCl. This plasmid inE. coli was deposited with the Centraal Bureau of Schimmelcultures(CBS), Baarn, the Netherlands, under the provisions of the BudapestTreaty on Dec. 12, 1986, under CBS 568.86. The construct is shown inFIG. 2.

B. Expression of hmulti-CSF in COS 1 Cells and Bioassays

pLB4 DNA was transfected to COS 1 cells using the calcium phosphatecoprecipitation method (Wigler et al., 1978). Cells were cultured for48-72 hours in alpha medium containing 10% fetal calf serum. The culturemedium was recovered, filtered, and used in assays for establishing itsbiologic activity. Human bone marrow progenitor colony assays and acutemyeloid blasts colony and proliferation assays were performed asfollows. Bone marrow was obtained from hematologically normal adultvolunteers by posterior iliac crest puncture following informed consent.The mononucleated cells were separated by density gradientcentrifugation on a Ficoll gradient (Nijegaard and Co., Oslo, Norway),washed and resuspended in Hank's balanced salt solution (HBSS). Myeloidcells and T-lymphocytes were then removed. For this purpose, marrowcells were lysed following incubation with monoclonal antibodies OKT-3(CD3; Ortho, Ravitan, N.Y.) and Vim 2 (myelo-monocytic cells; Majdic etal., 1984) at saturating concentrations in the presence of rabbitcomplement (40%; 30 minutes, 25° C.) according to established procedures(Lowenberg and Bauman, 1984). The cells were washed two times in HBSS,resuspended in Iscove's modified Dulbecco's medium (IMDM) and culturedin the presence of autologous plasma according to Fauser and Messner(1978), as described before (Delwel et al., 1986), at a concentration of1.5-3×10⁴ /ml. Erythropoietin 1 U/ml (sheep, step III, Connaught,Willowdale, Canada) and COS/pLB4 CM were added as growth-stimulatingactivities. Results of standard cultures withphytohemagglutinin-stimulated leukocytes CM (PH-LCM) in directcomparison with CS/pLB4 CM are also given. Sixty percent of the colonieswere plucked and identified by microscopic analysis. The CM from COScells transfected with the vector without insert (pLO) failed tostimulate colony formation by itself.

The results are shown in FIG. 3. As shown in the figure, the meannumbers of erythroid (BFU-E), granulocyte-macrophage (CFU-GM),granulocyte (CFU-G), eosinophil (CFU-Eo), macrophage (CFU-M) and mixed(CFU-MIX) colonies (±SD) are shown of duplicated cultures stimulatedwith graded volumes of COS/pLB4 CM.

Induction of AML Proliferation (see FIG. 4)

AML blasts were purified using a bovine albumin (BSA) density gradient.Residual T-lymphocytes were removed from the AML samples by E rosettesedimentation (Lowenberg et al., 1980; Swart et al., 1982; Swart andLowenberg, 1984). AML (patient 1) colony formation was determined notonly in the established PHA leukocyte feeder (PHA 1.f) system, but alsoin a modified version of the technique in which the leukocytes werereplaced by COS/pLB4 CM, permitting assessment of its colony-stimulatingactivity (Lowenberg et al., 1980; Lowenberg et al., 1982; Swart et al.,1982; Swart and Lowenberg, 1984) as shown in FIG. 4A. All experimentswere performed in triplicate. DNA synthesis of AML blasts (patient 2)was assayed by thymidine uptake as described (Touw et al., 1986) withresults shown in FIG. 4B. Both assays showed a dose dependentrelationship to COS/pLB4 CM added. Addition of control COS medium didnot affect AML proliferation in either assay.

C. Construction of Eukaryotic Expression Vector pLB4/BPV

In order to establish stable cell lines expressing human IL-3, C127cells (ATCC CRL 1616) were transfected with a derivative of pLB4. Thisderivative was constructed by insertion of the entire BPV-1 genome (Chenet al., 1982) into pLB4 by the following strategy. The BPV-1 BamHIfragment was excised from the vector pdBPV-MMTneo(342-12) (Law et al.,1983). The BamHI sticky ends were filled in using Klenow polymerase.Then the vector pLB4 was cleaved at the unique EcoRV site within the Ecogpt gene. Subsequently, the blunt-ended BPV-1 fragment was cloned intothe EcoRV-cleaved pLB4, resulting in the vector pLB4/BPV, which is ableto replicate in C127 cells. pLB4/BPV was transfected to C127 cells usingthe calcium phosphate precipitation method (Wigler et al., 1978). Thetransfected cells were cultured for 16 days, after which foci werepicked from the culture dishes. Several independent cell lines wereestablished. The pLB4/BPV vector appears to be stably maintained withinthe cells, as judged by Southern blotting of Hirt extracts (Hirt, 1967)of several cell lines. Conditioned culture medium was tested for IL-3activity using the AML proliferation assay. The stable cell linesproduce active human IL-3.

D. Construction of Eukaryotic Expression Vectors pGB/IL-328, pGB/IL-329and pGB/IL-330

An expression vector with elements similar to those in p8-4 of Sarver etal. (1985) was constructed. This vector contains a pML2 sequence(BamHI-ClaI, 2623 bp) and the Moloney murine sarcoma virus (MSV)enhancer fused to the mouse metallothionein I (MT) promoter. This fusionis described by Sarver et al. (1985). The vector also includes the SV40polyadenylation signal, as in Sarver et al. (1985), and the completeBPV-1 genome.

To construct the desired hIL-3 expression vector, the hIL-3-encodingAvaII-AvaI fragment from pLB4 (with the AvaI site filled in by Klenowpolymerase) was cloned into EcoRI/SmaI-cleaved pTZ18R (Pharmacia),together with a synthetic DNA fragment composed of the following twooligonucleotides: ##STR1## The resulting plasmid, pGB/IL-327, wassubsequently cleaved with BglII and BamHI, and the hIL-3-encodingfragment was isolated and cloned into the unique BglII site of theexpression vector described above. The resulting vector was namedpGB/IL-328. In this new hIL-3 expression vector, pGB/IL-328, the BglIIrecognition sequence is followed by 4 A-residues after which the ATGinitiation codon is placed. Use of the AvaI site in the 3' noncodingregion of the cDNA sequence for construction of the new expressionvector eliminates ATTTA repeats which have been implicated in mRNAinstability (Shaw and Kamen, 1986).

pGB/IL-328 was introduced into C127 cells as described above. Stablecell lines were established which produce high levels of humaninterleukin-3. These cell lines have a more than 20-fold higherproduction level of hIL-3 than stable cell lines derived from C127 cellscarrying pLB4/BPV. These cell lines allow for the efficient productionof hIL-3 with mammalian-type (complex) glycosylation. pGB/IL-328 wasalso introduced into FR3T3 cells (Seif and Cuzin, 1977) using themethods described above. Stable cell lines were also established,although with lower frequency. The obtained cell lines showed lowerhIL-3 productivity than the C127 cell lines transformed with pGB/IL-328.

pGB/IL-328 was also introduced into CHO cells (Wood and Burki, 1984)using the method described earlier. Since CHO cells do not form fociupon transfection of a vector carrying the BPV-1 sequences, aco-transfection with pSV2neo (Southern and Berg, 1982) was performed ina ratio of 10:1 (pSV2neo being the minor component), enabling selectionfor G418 resistance. G418-resistant colonies were isolated and testedfor IL-3 production. A significant amount of these clones producedhIL-3. Subsequently, a single cell cloning procedure was carried out inorder to establish stable cell lines. Several cell lines wereestablished with production levels equivalent and better than the levelof the best producing C127-pGB/IL-328 lines. This unexpected result hasno precedent in the prior art.

The cytomegalovirus IE enhancer/promoter combination was used forexpression of hIL-3. The 815 bp Bal-SphI fragment from plasmid pES (Boomet al., 1986) was cloned into SmaI-SphI cleaved pTZ18R (Pharmacia).Subsequently a BglII site was introduced just downstream of thetranscription initiation site using site-directed mutagenesis with thefollowing oligonucleotide: ##STR2##

An expression vector, pGB/IL-329, was constructed containing the pML2sequence from pGB/IL-328 (BamHI-EcoRI), the CMV enhancer/promoter(EcoRI-BglII) and the SV40 polyadenylation signal (BglII-BamHI) as inSarver et al. (1985). The final IL-3 expression construct pGB/IL-330 wasmade by introduction of the IL-3 encoding BamHI-BglII fragment frompGB/IL-327 into the unique BglII site of pGB/IL-329, followed byintroduction of the BamHI cleaved BPV-1 genome into the unique BamHIsite of this expression vector.

After introduction of pGB/IL-330 into C127 cells stable cell lines wereestablished which produced high levels of IL-3. Expression is 2-4 timeslower than with the pGB/IL-327 construct, however, pGB/IL-330 gives riseto mRNA with an extremely short 5' untranslated region of 12nucleotides. The introduction of a larger 5' untranslated region mayfacilitate translation, thereby enhancing the production level.

pGB/IL-330 was also introduced into CHO cells (Wood and Burki, 1984)using the co-transfection method described above. G418-resistantcolonies were picked and tested for hIL-3 production. Stable cell lineswere established by single cell cloning of the positive clones.Unexpectedly the best producing cell lines were equally productive asthe best CHO-pGB/IL328 lines indicating that either the short 5'untranslated region of the mRNA has no effect on the production of hIL-3or that the CMV enhancer/promoter is more effective in the CHO cells.

The IL-3 present in the conditioned media of the established cell linesshowed high activity in the biological assays described in WO 88/04691.

EXAMPLE 3 Construction of E. coli Expression Vectors A. Construction ofpGB/IL-301 (see FIGS. 5, 6, 7 and 8)

For construction of E. coli expression vectors, the followingmodifications were performed according to standard procedures (Maniatiset al., 1982).

1. The 3'-terminal noncoding sequences between the AvaI site (position541) and the XhoI site (position 856) in pLB4 were deleted by fusion ofthe DNA fragments following filling of the sticky ends with Klenowenzyme (FIG. 5).

2. For introduction of the hmulti-CSF insert into a bacterial expressionvector, the following steps were performed. The pLHl vector was digestedwith AvaII and the recessed ends filled with Klenow polymerase.Following ligation of a BglII linker (CAGATCTG), the DNA was digestedwith BglII and BamHI. The BglII-BamHI hmulti-CSF fragment was purifiedon polyacrylamide gel and subcloned in the BglII site of pTl, aderivative of pTZ18R (Pharmacia) modified in the multiple cloning site(see FIG. 6). Two clones were obtained, which had the insert in theopposite orientation with respect to the lacZ promoter (see FIG. 5).Inserts of these two clones were isolated on polyacrylamide gelfollowing digestion with BglII and EcoRV and subcloned in pTl digestedwith BglII and HindII.

The junction of the BglII linker and the hmulti-CSF DNA was verified bysequence analysis and showed a fusion of the linker to the AvaII sitelocated at nt 1 of the cDNA clone (this AvaII site had arisen byligation of the EcoRI linker to the cDNA molecule). Since this construct(pGB/IL-300) was not in phase with the lacZ protein, the BglII-EcoRVinsert was subcloned into BamHI- and HindII-digested pUC8 (Vieira andMessing, 1982). The resulting construct (pGB/IL-301, see FIGS. 5, 7 and8) was tested for production of a lacZ/hmulti-CSF fusion protein.

B. Construction of pGB/IL-302, pGB/IL-303, pGB/IL-304 and pGB/IL-305(FIGS. 5, 7 and 8)

Several base changes were introduced into the coding sequence for theN-terminal part of the fusion proteins by introduction of syntheticoligo nucleotides into pGB/IL-300. The new expression vectors, calledpGB/IL-302, pGB/IL-303, and pGB/IL-304, were constructed as follows. TheHindII-HindIII fragment of pGB/IL-300 was isolated on agarose gel andligated to a synthetic oligonucleotide comprising the nucleotides 99-137of hmulti-CSF and a 5' terminal SalI recognition sequence and insertedinto pTZ18R digested with SalI and HindIII. The sequence of severalclones was established. Indeed, several base changes were observed,resulting in modifications of the hmulti-CSF protein. Inserts of severalclones were transferred to pUC8 for expression of the lacZ fusionprotein (pGB/IL-302, pGB/IL-303). Clone pGB/IL-304 was made in phasewith lacZ by ligation of the SalI site following filling of recessedends with Klenow. Construction was verified by PvuI digestion. Severalclones lacked a synthetic oligonucleotide and were found to be fused inframe to the lacZ protein. One example of these clones was calledpGB/IL-305.

C. Construction of pGB/IL-306 (see FIGS. 5, 7 and 8)

An expression vector coding for a protein lacking the lacZ N-terminalamino acids was made from pGB/IL-300 by deletion looping as described inOsinga et al., 1983 The synthetic oligonucleotide comprised 22nucleotides upstream of the pTZ lacZ gene including the ATG start codonand the first 24 nucleotides coding for mature IL-3. This plasmid wascalled pGB/IL-306 (FIGS. 5, 7 and 8).

E. coli strains containing the plasmids pGB/IL-300, pGB/IL-301 andpGB/IL-302 were deposited with CBS on Jul. 13, 1987, under CBS 377.87,CBS 378.87 and CBS 379.87.

FIG. 8 shows the sequence of fusion regions for the various plasmidsconstructed. The sequence of the clones is given from the start of thelacZ protein coding region in either pUC8 or pTZ18R (lower case letters)and of the hmulti-CSF coding region (upper case letters) up to the ClaIsite at position 158. Mutations in the hmulti-CSF DNA sequence areunderlined, resulting in Trp¹³ →Arg¹³ (pGB/IL-302); Leu⁹ →Pro⁹ and Trp¹³→Arg¹³ (pGB/IL-303); Met³ →Thr³ and a silent change (pGB/IL-304).

In the parent application, EP 87201322.2, filed on Jul. 13, 1987, otherdesignations were used for these plasmids as follows:

pGB/IL-300=pT-hIL3;

pGB/IL-301=pUC/hmulti;

pGB/IL-302=pUC/hmultiDELTA1A;

pGB/IL-303 =pUC/hmultiDELTA1B;

pGB/IL-304=pUC/hmultiDELTA1C;

pGB/IL-305=pUC/hmultiDELTA2;

pGB/IL-306=pTZ/hmulti;

D. Expression of lacZ/hmulti-CSF Fusion Proteins and Nature hmulti-CSFin E. coli

E. coli strains (JM 109) carrying various expression vectors were grownin LB medium containing 50 ug/ml of ampicillin at 37° C. until anoptical density of 0.5 at 550 nm was reached. Subsequently IPTG(isopropyl beta-D-thiogalactoside, Pharmacia) was added to the cultureto a final concentration of 1 mM and incubation was continued for 3-4hours.

Plasmids pGB/IL-306 and pGB/IL-302 were also transformed to E. coli DH1(wild-type lacZ operon). Those strains were grown in LB medium or 2×TYmedium containing 50 ug/ml of ampicillin at 37° C. for 16 hours.

Bacteria were collected by centrifugation and sonicated in buffercontaining 0.1M Tris/HCl, pH 8.0; 5 mM EDTA 0.2% Nonidet P40 (NP-40) and1 mM phenylmethylsulfonyl fluoride (PMSF) and centrifuged for 30 min at20,000× g. Polyacrylamide gel electrophoresis of the pellet andsupernatant fractions showed that the bulk of the hmulti-CSF protein isstored in the bacteria in an insoluble form.

The pellet was reextracted with 0.5% NP-40 buffer and finallysolubilized with 8 M urea 0.1M Tris/HCl, pH 8.0 and 5 mM dithiothreitol.Thus, an extensive purification of the fusion proteins was achieved(FIG. 9).

As shown in the figure, inclusion bodies from bacteria (E. coli)containing pGB/IL-301 and pGB/IL-302 were isolated as described. Lanes 1show the 0.2% NP40 supernatant (sample corresponds to 0.1 ml of theoriginal bacterial culture). Lanes 2 show the 0.5% NP40 supernatant (0.2ml) and lanes 3 the pellet solubilized in 8M urea buffer (A: 0.05 ml; B:0.2 ml). The proteins were separated on a 13.5% SDS-polyacrylamide geland stained with Coomassie Brilliant Blue. Molecular weights (in kd) ofmarker proteins (lane M) are denoted on the right. The human multi-CSFfusion proteins are indicated by arrows. The fusion protein encoded bypGB/IL-301 has a molecular weight, as expected, of about 20 kd; thatproduced from pGB/IL-302, of about 16 kd.

E. Determination of Biological Activity of Bacterial hmulti-CSFPreparations

Bacterial protein preparations were diluted in alpha medium containing1% bovine serum albumin, filter sterilized and assayed in the AML blastproliferation assay. Diluted samples were added to purified AML blastsand cultured for four days. DNA synthesis was measured using ³ Hthymidine as described (Touw et al., 1986). One unit per ml is definedas the amount of hmulti-CSF required for half maximal proliferation ofAML blasts. FIG. 10 shows this titration. Various dilutions of the ureaextracted protein preparation of bacteria containing the plasmidpGB/IL-302, were assayed for the stimulation of AML blast proliferationusing ³ H-thymidine. The fusion protein concentration of this proteinpreparation was 33 ug/ml. Based on the presented titration curve, theactivity of this preparation is 16,000 units/ml.

The amount of bacterial fusion protein in the preparations was estimatedfrom polyacrylamide gel electrophoresis and used for calculatingspecific activities.

The results are shown in the following table:

                  TABLE 1                                                         ______________________________________                                        Biological Activity of Bacterial hmulti-CSF Preparations                                                          Specific                                           Mr (× 10.sup.-3)                                                                 ug protein                                                                              Units   activity                                           lacZ/hmulti                                                                            per ml    per ml  units per                                          (1)      (2)       (3)     mg IL-3                                   ______________________________________                                        pGB/IL-301 20         20        45     4,500                                  pGB/IL-302/303                                                                           16         5         2400  480,000                                 pGB/IL-304 18         ND(4)     18    --                                      pGB/IL-305 16         1         300   300.000                                 pGB/IL-306 15         ND        70    ND                                      ______________________________________                                         1. Approximate molecular weights are estimated from the DNA sequence of       the fusion protein (FIG. 8).                                                  2. IL3 concentrations were estimated on SDSpolyacrylamide gel and             calculated per ml of starting culture.                                        3. Activity of urea solubilized protein was determined in the AML             proliferation assay and is expressed per ml of starting culture.              4. Not determined.                                                       

From these results it was concluded that human multi-CSF expressed as afusion protein in E. coli was obtained in biologically active form. Theresults show that changes introduced into the N-terminus of the fusionproteins may influence the specific activity of these proteins.

EXAMPLE 4 Preparation of Antibody Preparations Capable of ImmunospecificReaction with Human IL-3 Protein A. Polyclonal Rabbit Anti-Human IL-3Antiserum

A preparative gel was made from a lysate of E. coli containing theplasmid pGB/IL-301. The 21 kd band with the IL-3 fusion protein wassliced out, minced in saline with a mortar and emulsified in a 1:1 ratioin Complete Freund's Adjuvant containing 1 mg of Mycobacteriumtuberculosis H37RA per ml. New Zealand White rabbits (spf) wereimmunized with 1 ml of the emulsion (with ±100 ug IL-3 fusion protein)divided over 5 injection sites (2×i.m. in the thighs, 3×s.c. on theback). Booster injections of the same antigen in Incomplete Freund'sAdjuvant were given at week 2, 4 and 6. Serum was collected at week 8 byvenapuncture from the ear.

One volume of serum was absorbed with 9 volumes of sonicated pUC8containing E. coli (overnight at 4° C.) to remove nonspecificantibodies. Immunoblotting of all IL-3 constructs made in E. coli, B.licheniformis, B. subtilis and S. cerevisiae, and K. lactis showedimmunospecific reaction with the absorbed sera at a dilution of 1 in6500.

Some of these results are shown in FIG. 11. The proteins were isolatedfrom the recombinant hosts as described above and were separated on a13.5% polyacrylamide gel and blotted onto a nitrocellulose membrane.Lane 1: E. coli containing pTZ18R (control); Lane 2: pGB/IL-301; Lane 3:pGB/IL-301; Lane 4: pGB/IL-302; Lane 5: pUC19 (control); Lane 6:pGB/IL-301; Lane 7: pGB/IL-302. Lanes 6 and 7 show proteins present inthe pellet after the sonication of the bacteria. Lanes 3, 4 and 5 showproteins present in the pellet after the first washing step. Lanes 1 and2 show the final urea-solubilized protein fractions.

The arrows show the fusion proteins (of the expected size) frompGB/IL-301 and pGB/IL-302.

FIG. 12A shows the inhibition of IL-3 dependent proliferation of AMLblast cells by anti-IL-3 antiserum. FIG. 12B shows that preimmune serumdoes not affect the action of IL-3 on AML blast cell proliferation. Inboth panels. =IL-3 at 10 U/ml; =IL-3 at 1 U/ml; =control no addition.

FIG. 12A shows IL-3 dependent growth in the AML blast proliferationassay (Touw et al., 1986) was inhibited by the sera in a dose dependentmanner; FIG. 12B shows preimmune sera do not have this effect. Ascontrol, GM-CSF dependent growth was unaffected by these sera in thesame assay (FIG. 12A where =GM-CSF at 100 U/ml.

B. Monoclonal Mouse Anti-Human IL-3 Antibodies

Balb/C mice were immunized with 3×0.1 ml (s.c.) of the same emulsion asused for the rabbits. A booster (0.1 ml i.p.) of antigen in IncompleteFreund's Adjuvant was given at week 2 and three days later spleenlymphocytes were fused with SP2/0 myeloma cells according to standardprocedures (Salfre and Milstein, 1981). Hybridoma supernates werescreened in the Enzyme Linked Immunosorbent Assay, using a lysate of E.coli pGB/IL-302 (containing the 17 kd IL-3 fusion product) as a positivecontrol and a lysate of E. coli pUC8 as negative control. In total, 29IL-3 hybridoma cultures secreting antibodies specific for IL-3 wereselected and stabilized.

EXAMPLE 5 Construction of Bacillus Expression Vectors

General cloning techniques were used (Maniatis et al., 1982).

A. Construction of pGB/IL-307 (FIG. 13)

For construction of pGB/IL-307 the SmaI fragment of pLB4 carrying thehmulti-CSF gene, was ligated into PvuII digested pUB110 (Gryczan et al.,1978). After transformation to competent cells (Bron and Venema, 1972)of DB105 (an spo⁻ derivative of the protease deficient strain DB104(Kawamura and Doi, 1984), two clones were obtained, as expected; thefragment was cloned in both orientations. The plasmid that harbored thefragment in the correct orientation with respect to the so-called "HpaIIpromoter" (Zyprian and Matzura, 1986) was called pGB/IL-307. In thiscase a fusion protein will be made (see FIG. 13).

B. Construction of pGB/IL-310

An hmulti-CSF expression plasmid was prepared as described below.

1. Promoter Cloning (FIG. 14)

For expression in Bacillus, a synthetic sigma-⁴³ promoter as described(EPA0024.294) is used (the promoter used to be called sigma-⁵⁵).

Plasmids pPROM55s (EPA0024.294), the promoter containing plasmid, andpGPA14 (EPA0244042) were digested with EcoRI and XbaI. The promoterfragment was ligated into the vector fragment, which had been purifiedon an agarose gel. After transformation to E. coli (JM 101), the correctplasmid was obtained and called pGB/IL-308 (FIG. 14).

2. Introduction of a Synthetic Oligonucleotide into pGB/IL-308 (FIG. 15)

A synthetic oligonucleotide comprising the nucleotides 39-158 and484-546 of hmulti-CSF, a 5'-terminal SalI recognition sequence- and a3'-terminal XmaIII site was ligated into SalI-XmaIII digestedpGB/IL-308. The ligation mixture was introduced into JM101. Afteranalysis of a number of transformants, the correct plasmid was found,pGB/IL-309.

3. Introduction of hIL3 (FIG. 16)

After transformation to and isolation from B. subtilis DB105, theplasmid pGB/IL-309 was digested with XmaIII. The recessed ends werefilled in with Klenow polymerase, and the plasmid then was cleaved withClaI. The plasmid pGB/IL-307 was digested with AvaI, the ends filled inwith Klenow and then digested with ClaI. Subsequently, the hmulti-CSFcontaining fragment was ligated with the pGB/IL-309 fragment andtransformed to JM101. The resulting plasmid was called pGB/IL-310 (FIG.16). This plasmid harbored the hIL-3 gene with its own signal sequence.After isolation of the correct plasmid, it was also introduced into B.subtilis DB105.

C. Construction of pGB/IL-311 and pGB/IL-312 (FIGS. 17, 18)

pGB/IL-310 was partially digested with HindIII and totally with PvuII.The two hmulti-CSF containing PvuII digests were subsequently digestedwith HindIII and SmaI.

FIG. 17 shows the nucleotide sequence of plasmid pBHA1. The plasmidconsists of positions 11-105 and 121-215; bacteriophage FD terminator(double): positions 221-307; a part of plasmid pBR322 (viz., positions2069-2153): positions 313-768; bacteriophage F1, origin of replication(viz., positions 5482-5943): positions 772-2571; part of plasmid pBR322,viz., the origin of replication and the beta-lactamase gene: positions2572-2685; transposon Tn903, complete genome: positions 2719-2772;tryptophan terminator (double): positions 2773-372-9; transposon Tn9,the chloramphenicolacetyltransferase gene. The nucleotides at position3005 (A), 3038 (C), 3302 (A), and 3409 (A) differ from the wild-type catcoding sequence. These mutations were introduced so as to eliminate theNcoI, BalI, EcoRI and PvuII sites: positions 3730-3804; multiple cloningsite: positions 3807-7264; part of plasmid pUB110, viz., the replicationfunction and kanamycin resistance gene (EcoRI-PvuII fragment) (McKenzieet al., 1986; McKenzie et al., 1987): positions 7267-7331; multiplecloning site. The fragments were put together by known cloningtechniques, e.g., filling in of sticky ends with Klenow, adaptercloning, etc. All data were derived from Genbank® National Nucleic AcidSequence Data Bank, NIH, USA.

After transformation to JM101 and analysis of a number of ampicillinresistant colonies, two different plasmids were found: pGB/IL-312, whichharbored the complete gene with complete control sequences, andpGB/IL-311, which contained the complete gene and the promoter lackingthe -35 region in the other orientation (see FIG. 18).

pGB/IL-311 has been transformed to B. subtilis DB105 and B.licheniformis strain T9 (DELTAamy, spo⁻, exoprotease negative, rif^(r),see EPA87201379.2).

D. Construction of pGB/IL-313 (FIG. 19)

In order to obtain a smaller plasmid, with the hmulti-CSF genedownstream of the "HpaII promoter", pGB/IL-312 was digested with BamHIand religated. The ligation mixture was transformed into competent DB105cells. A number of neomycin resistant colonies were analyzed and thecorrect plasmid was obtained. The plasmid was called pGB/IL-313.

E. Construction of pGB/IL-317 (FIG. 20)

In order to clone the hmulti-CSF gene downstream of the B. licheniformisalpha-amylase transcriptional and translational initiation region andsignal sequence, one of the earlier-described pOL5-delta vectors(EPA87201379.2) was used, viz., pOL5-2-delta. Besides the alpha-amylasesignal sequence (29 amino acids long), this plasmid harbors one aminoacid of the alpha-amylase mature sequence (an Ala) followed by amultiple cloning site: EcoRI-SmaI-SalI, HindIII (EPA87201379.2).

The SalI-PvuII fragment of plasmid pGB/IL-310 containing the hmulti-CSFgene was ligated into the SalI-PvuII-digested pOL5-2-delta vector andtransformed to DB105. The resulting plasmid was called pGB/IL-317 (FIG.20). The hIL-3 gene still harbors its own signal sequence on thisplasmid. The plasmid was also introduced into B. licheniformis T9.

F. Construction of pGB/IL-322 and pGB/IL-326

In order to obtain an improved Bacillus expression vector for humanIL-3, a derivative of plasmid pGB/IL-317 was made. This plasmid containsdownstream of the strong alpha-amylase promoter, in a 5'-to-3'direction, coding information for the Bacillus alpha-amylase signalsequence, extra amino acids, the IL-3 signal sequence, mature IL-3, andthe 3' end of the amylase gene.

First, a perfect junction between the DNA sequence encoding the amylasesignal sequence and the DNA sequence encoding mature human IL-3 was madein the E. coli- Bacillus shuttle-plasmid pGB/IL-311. This plasmid wascleaved with restriction enzymes SalI and ClaI. subsequently, the smallfragment containing the information for the human IL-3 signal sequencewas exchanged with a synthetic SalI-ClaI fragment containing the codinginformation for the alpha-amylase signal sequence fused to the matureIL-3 coding sequence. The resulting plasmid, pGB/IL-321, was transformedto B. subtilis 1A40 and to B. licheniformis T9. Transformants synthesizeonly small amounts of IL-3, but the protein is secreted into the medium.

The small PstI-ClaI fragment from pGB/IL-321 was isolated and insertedinto the PstI-ClaI-cleaved plasmid pGB/IL-317, thereby exchanging theDNA sequence encoding the extra amino acids and the IL-3 signal sequencewith the perfect junction of alpha-amylase signal sequence and matureIL-3 coding sequence. The resulting plasmid was designated pGB/IL-322.pGB/IL-322 gives rise to a high production of IL-3 by B. licheniformisT9 transformants. This was not determined for B. subtilis 1A40. Morethan 95% of the protein is secreted into the culture medium. N-terminalsequencing of the purified protein showed that the IL-3 synthesized bythese transformants has the correct amino-terminus (Ala¹ -Pro² -Met³ . .. ).

Another new expression vector, pGB/IL-326, was constructed as follows.The HindIII-SalI fragment (HindIII end filled in using Klenowpolymerase) from pGB/IL-311 was cloned into pTZ18R (Pharmacia) cleavedwith SalI and SmaI, resulting in vector pGB/IL-323. Subsequently, theDNA encoding the IL-3 signal sequence was exchanged with a synthetic DNAfragment encoding the Bacillus alpha-amylase signal sequence. In thissynthetic piece of DNA, the ATG start codon is preceded by the sequenceCAT resulting in a cleavage site for the restriction enzyme NdeI. TheSphI-KpnI fragment from this plasmid pGB/IL-324, containingalpha-amylase signal sequence, mature IL-3, and amylase terminator, wasthen cloned into pBAH3 cleaved with SphI and KpnI (pBAH3 is a derivativeof pBAHl lacking the PstI site in the Ap^(R) gene). The resultingplasmid, pGB/IL-325, was subsequently cleaved with NdeI and religated,thereby fusing the so-called HpaII promoter with the alpha-amylasesignal sequence and the mature IL-3 encoding sequence. The resultingplasmid, pGB/IL-326, was transformed to B. licheniformis T9.Transformants produced high amounts of IL-3 of which more than 95% wassecreted into the culture medium. For proper secretion of mature hIL-3by Bacillus, a perfect junction between alpha-amylase signal sequenceand mature hIL-3 coding sequence appears crucial.

The mature IL-3 produced by B. licheniformis T9 transformants obtainedwith the expression vectors pGB/IL-322 and pGB/IL-326 has the correctamino acid sequence, is unglycosylated, and shows high biologicalactivity both in AML and human bone marrow assays. Thus, surprisingly,by rearrangement of the different genetic elements with respect to eachother, optimal combinations of promoters, signal sequences and maturehIL-3 coding sequences were found. pGB/IL-326 was also transformed to B.subtilis 1A40.

G. Expression of Eight Expression Plasmids in Bacillus Strains

B. subtilis and B. licheniformis strains carrying the expressionplasmids mentioned below were grown in TSB medium containing 20 ug/mlneomycin or 10 ug/ml erythromycin at 37° C. (for 16-24 hours); 300 ug/mlof the culture was centrifuged. The pellet was resuspended in samplebuffer and analyzed using polyacrylamide gel-electrophoresis followed byWestern blotting. The supernatant was TCA precipitated, and the pelletwas resuspended in sample buffer. Both supernatant and pellet wereanalyzed for IL-3 protein (see Table 2).

To determine the biological activity of the produced proteins, thefollowing steps were carried out: the cell pellets were resuspended in abuffer containing 0.1M Tris/HCl pH 8.0 and 10 mM MgCl₂. Lysozyme wasadded to a final concentration of 1 mg/ml and PMSF to a finalconcentration of 1 mM. The solution was incubated for 30 min. at 37° C.Subsequently DNase (final concentration 20 ug/ml) was added and thesolution was incubated for 15 min. at 20° C. Finally, the biologicalactivity of this preparation as well as of the supernatant of thecultured cells was determined as described. The results are shown inTable 2.

                  TABLE 2                                                         ______________________________________                                        Expression of the Bacillus Vectors                                                       MW IL-3     Biological                                                        Pellet Supernat.                                                                              Activity                                           Plasmid  Strain  (kd)     (kd)   Pellet                                                                              Supernat.                              ______________________________________                                        pGB/IL-307                                                                             DB105   21       --     +     -                                      pGB/IL-310                                                                             DB105   15; 17   15; 17 -     -                                      pGB/IL-311                                                                             DB105   12.5; 15 --     +     -                                               T9      --       --     +     -                                      pGB/IL-313                                                                             DB105   15; 17   12.5; 15                                                                             +     -                                               T9      --       --     +     -                                      pGB/IL-317                                                                             DB105   12.5; 15 12.5; 15                                                                             +     +                                                       17; 20   17                                                           T9      12.5; 15 12.5; 15                                                                             +     +                                                       17; 20   17                                                  pGB/IL-321                                                                             1A40    --       --     -     +                                               T9      --       --     -     +                                      pGB/IL-322                                                                             1A40    N.D.     N.D.   N.D.  N.D.                                            T9      14.5; 17 14.5   +     +++++                                  pGB/IL-326                                                                             1A40    14.5; 17 14.5   +     ++                                              T9      14.5; 17 14.5   +     +++++                                  ______________________________________                                    

It can be concluded that in B. subtilis, using pGB/IL-307, a fusionprotein is made that has IL-3 activity. When the human IL-3 gene onlycontains its own signal sequence, no significant secretion of human IL-3is obtained. All IL-3 activity is found intracellularly. In those casesit seems that besides precursor IL-3, mature IL-3 (15 kd) has beenformed in the cell. Thus, some transport across the membrane might havetaken place, but the protein is not transported across the cell wall.However, using the alpha-amylase regulation and secretion signals(pGB/IL-317), most of the IL-3 activity appeared to be secreted into theculture medium. Besides a degradation product, two proteins are detectedin the supernatant, one of about 15 kd and one of about 17 kd, mostprobably mature IL-3 and precursor IL-3, respectively. These dataindicate that both processing sites, viz., the alpha-amylase and thehmulti-CSF processing site, are used. In the cell the most abundantproduct is precursor IL-3 containing the alpha-amylase signal sequence(the 20 kd protein) as shown by Western blotting. Sometimes adegradation product is detected.

EXAMPLE 6 Construction of Kluyveromyces Lactis Expression Vectors A.Construction of pGB/IL-316

A DNA fragment comprising the Tn5 gene (Reiss et al., 1984) conferringresistance to gentamycin G418, under the direction of the alcoholdehydrogenase I (ADHI) promoter from S. cerevisiae, similar to thatdescribed by Bennetzen and Hall (1982), was inserted into the SmaI siteof pUC19 (Yanisch-Perron et al., 1985). An E. coli strain containing theobtained plasmid, pUC-G418, was deposited with CBS on Dec. 4, 1987,under CBS872.87.

Into the XbaI-HindIII cleaved pUC-G418 vector an XbaI-HindIII fragmentfrom plasmid pGB903 (U.S. patent application Ser. No. 07/078,539)containing the K. lactis lactase promoter and calf prochymosin DNA wasinserted, resulting in plasmid pGB/IL-314.

The SalI-HindIII fragment from this plasmid was replaced by a syntheticDNA fragment containing a small multiple cloning site and the lactaseterminator (see FIGS. 21, 22). The resulting plasmid is designatedpGB/IL-315.

In the SacII-XhoI cleaved pGB/IL-315 vector the following fragments wereligated:

1. The SacII-XbaI fragment from pKS105 (U.S. patent application Ser. No.07/078,539; U.S. patent application Ser. No. 07/078,539) carrying the 3'part of the lactase promoter and the 5' part of the alpha-factor signalsequence of S. cerevisiae.

2. A synthetic oligonucleotide comprising the 3' part of thealpha-factor signal sequence starting at the XbaI site and the 5' partof the mature hIL-3 cDNA sequence up to the 5' half of the HpaI site(amino acid residue 14).

3. The HpaI-XhoI fragment carrying most part of the hIL-3 cDNA sequence(residue 15-133 plus the 3' noncoding region). The resulting plasmid,designated pGB/IL-316, is depicted schematically in FIG. 21. Thecomplete vector sequence from the SacII site in the lactase promotersequence up to the HindIII site at the end of the synthetic terminatoris given in FIG. 22.

FIG. 22 shows the nucleotide sequence of plasmid pGB/IL-316 between theunique SacII site in the lactase promoter and the HindIII site behindthe terminator (residues 4457 to 7204). Residues 4457 to 6100 compromisethe lactase promoter sequence. Residues 6101 to 6355 compromise thealpha factor signal sequence. Residues 6356 to 7115 compromise thesequence for mature human IL-3 plus the 3' noncoding cDNA sequence.Residues 7116 to 7204 compromise the synthetic terminator sequence.

B. Construction of pGB/IL-318

An expression vector similar to pGB/IL-316 was constructed in which thecoding information for the alpha factor signal sequence of S. cerevisiaewas replaced by the alpha-factor signal sequence of K. lactis (U.S.patent application Ser. No. 07/078,539). The remaining part of theplasmid is identical to pGB/IL-316. The sequence of pGB/IL-318 betweenthe SacII site in the lactase promoter and the HindIII site behind theterminator (residues 4457 to 7190) is given in FIG. 23.

Residues 4457 to 6087 comprise the sequence of the lactase promoter anda small linker sequence. Residues 6088 to 6342 comprise the K. lactisalpha factor signal sequence. Residues 6343 to 7102 comprise thesequence for mature human IL-3 plus the 3' noncoding cDNA sequence.Residues 7103 to 7190 comprise the synthetic terminator sequence.

C. Transformation of Kluyveromyces Lactis and Analysis of Secreted hIL-3

Plasmid pGB/IL-316 and pGB/IL-318 was digested at the unique SacII sitein the lactose promoter region, and used to transform K. lactis strainCBS 2360 (see U.S. patent application Ser. No. 07/078,539). Integrationof the plasmids is thus targeted to the chromosomal lactase genepromoter region. The resulting G418-resistant transformants were grownto saturation in liquid YEPD medium, and the culture supernatants andcell lysates were assayed for IL-3 activity using the AML cell DNAsynthesis assay.

Virtually all IL-3 appeared to be secreted into the culture medium, andto be active. The proteins from the culture supernatant wereprecipitated using ethanol and analyzed using denaturing polyacrylamidegel electrophoresis followed by Western blotting. The predominantproduct has an apparent MW of about 21 kd, whereas also a distinct bandat about 15 kd is observed. The latter product most probably correspondsto the mature unglycosylated IL-3, whereas the 21 kd product is theproduct carrying core glycosylation at the two potential glycosylationsites. Incubation with Endoglycosidase H results in a protein migratingin the 15 kd range, suggesting that all IL-3 is processed correctlyduring the secretion process and that the bulk of the protein is beingglycosylated.

EXAMPLE 7 Construction of a Saccharomyces Cerevisiae Expression VectorA. Construction of pGB/IL-319

First an expression vector called pGB/TEFact was constructed On thispTZ18R (Pharmacia)-derived plasmid the S. cerevisiae translationelongation factor (EF-1alpha) promoter sequence which was cloned andsequenced as described (Najata et al., 1984; Nagashima et al., 1986), iscoupled by means of a small SalI-BglII-XhoI linker to the S. cerevisiaeactin transcription terminator sequence (Gallwitz and Sures, 1980),which was synthesized using an Applied Biosystems DNA synthesizer. Thesequence of the expression cassette is given in FIG. 24. Residues 1 to949 comprise the EF-1alpha promoter. Residues 950 to 967 comprise thesequence of the SalI-BglII-XhoI linker. Residues 968 to 1113 comprisethe actin terminator sequence.

The unique SmaI site in pGB/TEFact was used to introduce the G418resistance cassette described in Example 6. The resulting plasmid wascalled pGB/TEFactG418.

Finally, the hIL-3 expression vector pGB/IL-318 was constructed byintroduction of the following DNA sequences into the SalI-XhoI-cleavedpGB/TEFactG418 plasmid:

1. The SalI-NruI fragment from pGB/IL-316 carrying the S. cerevisiaealpha factor signal sequence and the hIL-3 coding sequence up to theNruI site.

2. A synthetic NruI-XhoI DNA fragment comprising the remainingnucleotides coding for hIL-3 and the XhoI recognition sequenceimmediately following the TGA stop codon.

B. Transformation of Saccharomyces Cerevisiae and Analysis of SecretedhIL-3

Plasmid pGB/IL-319 was cleaved at the unique EcoRI site in the EF-lalphapromoter. Integration of the plasmid is thus targeted to the chromosomalEF-1alpha region. S. cerevisiae wild-type strain D273-103 (alpha; ATCC25657) was transformed as described for K. lactis (U.S. patentapplication Ser. No. 07/078,539). The G418-resistant colonies werepicked, and transformants were given to saturation in liquid YEPDmedium. The culture supernatant was assayed for hIL-3 activity using theAML assay. The protein produced by S. cerevisiae was found biologicallyactive.

The proteins from the supernatant were precipitated using ethanol andsubsequently analyzed by polyacrylamide gel electrophoresis followed byWestern blotting. Two prominent products could be distinguished on theWestern blot, a 21 kd glycosylated product and an unglycosylated productof about 15 kd.

EXAMPLE 8 Construction of Mammalian Expression Vectors

The BPV genome encodes 3 proteins with sequences of the E2 open readingframe called E2-ta, E2-tr and E8/E2 respectively (Lambert et al., 1988).The ATG codon at position 3091-3093 may be changed into either an GCG(Ala) or an ACG (Thr), thereby on the one hand prohibiting thetranslation initiation of the E2-tr protein, and on the other hand,substituting Met¹⁶² in E2-ta with Ala¹⁶² or Thr¹⁶². The BPV genome inpGB/IL-328 and pGB/IL-330 may be exchanged by the E2-tr mutant BPVgenomes. The equivalents of pGB/IL-328 are designated pGB/IL-331 (Ala)and pGB/IL-332 (Thr). The equivalents of pGB/IL-330 are designatedpGB/IL-333 (Ala) and pGB/IL-334 (Thr). The new expression vectors can beintroduced into C127 and CHO cells as described. The level of hIL-3production is expected to be substantially raised in both cell typeswith respect to the C127-pGB/IL-328, CHO-pGB/IL-328 and C127-pGB/IL-330, CHO-pGB/IL-330 combinations respectively.

The IL-3 present in the conditioned media of the established cell lineswill show high activity in the biological assays described in WO88/04691.

EXAMPLE 9 Purification of hIL-3 from Heterologous Expression Systems A.Purification of hIL-3 from Bacillus licheniformis T9 (pGB/IL-322)

In order to obtain highly purified and homogeneous hIL-3 from Bacilluslicheniformis T9 containing plasmid pGB/IL-322, isolation procedureswere developed that offer extended possibilities for upscaling.Cell-free medium from B. licheniformis T9 was brought to 1M (NH₄)₂ SO₄and adjusted to pH 7.0 with NaOH and loaded on a column of FractogelTSK-Butyl 650C, equilibrated in 1M (NH₄)₂ SO₄ in 10 mM Tris-HCl buffer,pH 7.0. One mM phenylmethylsulfonylfluoride (PMSF) was used as aproteinase inhibitor. Whereas most of the protein was found in therun-through fractions, hIL-3 was adsorbed to the column. After extensivewashing of this column with the same buffer, hIL-3 was eluted in agradient from 1M to 0M (NH₄)₂ SO₄ in 10 mM Tris-HCl, pH 7.0. ThehIL-3-enriched fractions were concentrated by (NH₄)₂ SO₄ precipitationat 70% saturation, and partly desalted by dialysis against water (up toa conductivity that was identical or lower than the conductivity of 20mM Tris-HCl, pH 7.8) and loaded at pH 7.8 on a column of FractogelTSK-DEAE 650M, equilibrated in 20 mM Tris-HCl, pH 7.8. Again, most ofthe hIL-3, now found in the run-through fractions, could be separatedfrom the contaminating protein that remained bound to the column. For afinal isolation procedure, the hIL-3-containing solution wasconcentrated (either by adsorption and separation on a small column ofFractogel TSK-Butyl 650C as described above, or by (NH₄)₂ SO₄precipitation) and purified to homogeneity by gel filtration on Biogel A(0.5M; 100-200 mesh) with 200 mM NaCl in 20 mM Tris-HCl, pH 7.0, as therunning buffer. hIL-3 obtained at this stage of the purificationconsisted of several hIL-3 degradation products and was free fromcontaminating proteins, as was determined by SDS-PAGE followed by silverstaining and by immunoblotting using anti-hIL-3 antibodies. The observeddifferent molecular forms of hIL-3 most probably are the result ofproteolytic degradation of the proteins during the fermentationprocedure.

Further analysis of this preparation of hIL-3 resulted in singleA280-detectable peak elutions after separation by reverse-phase HPLC(using a gradient from 40-60% acetonitrile in 0.1% trifluoroacetic acid,TFA), gel filtration on an HPLC TSK G2000SW column (7.5×600 mm) andcation-exchange chromatography on Fractogel TSK CM 650M. (IL-3 wasloaded at pH 5.0 and eluted in a phosphate-buffered gradient from pH 5.0to pH 9.0.)

If hIL-3 degradation products were present in the starting mediumobtained from B. licheniformis T9, selective removal of theseproteolytic degradation products from the fraction of interest wascarried out by the following purification procedures.

hIL-3 enriched fractions derived from the hydrophobic interaction columnabove were concentrated as described, adjusted to pH 7.8 and brought toa conductivity of 0.7 mS. hIL-3 was then loaded on a column of QSepharose Fast-Flow (50×5 cm) that was equilibrated in a Tris bufferwith identical pH and conductivity (with a flow of about 300ml/h). Inthis purification step, the volume of the concentrated solution withhIL-3 that was loaded did comprise just 3-4% of the total column volume.All active forms of hIL-3 were found in the run-through fractions. Themajor form of hIL-3 was separated from other hIL-3-like smaller proteinsthat were also positive in western blotting using antihuman IL-3antibodies. When contaminating hIL-3 degradation products were stillpresent in the peak fraction of interest, this isolation procedure wasrepeated. Amino-terminal sequence analysis of the first 30 amino acidsof the major form of hIL-3 revealed a sequence that is identical withthe mature protein.

Chromatography on hydroxylapatite also turned out to be an effectivemethod for the isolation of several B. licheniformis hIL-3 degradationproducts. hIL-3 was loaded on a Biogel hydroxylapatite column in 10 mMNaH₂ PO₄, 0.01 mM CaCl₂, 0.02% NaN₃, pH 6.8, and was eluted from thecolumn in a gradient from 10 to 350 mM NaH₂ PO₄ in the same solution.This method was effective on both an analytical and a preparative scale.All hIL-3 preparations obtained were positive in the AML assay asdescribed herein.

hIL-3 degradation products from B. licheniformis T9 could also beseparated by means of chromatofocussing on the polybuffer exchangerpBE94. By the application of a pH-gradient onto the column, differenthIL-3 degradation products were eluted between pH 6.5 and pH 8.5.

A similar isolation procedure based on hydrophobic interaction of hIL-3from B. licheniformis on octyl-Sepharose instead of Fractogel TSK-Butyl,was performed. hIL-3 was eluted by the application of a gradient from 0%(v/v) to 100% (v/v) ethylene glycol which illustrates the strongerbinding characteristics of the former matrix.

B. Large-Scale Purification of hIL-3 from Bacillus licheniformis T9(pGB/IL-322)

hIL-3 was purified from B. licheniformis T9 (pGB/IL-322) using thefollowing four step large-scale purification scheme.

Step One: Hydrophobic Interaction Chromatography

The cell-free supernatant derived from 6 fermentation runs of B.licheniformis T9 (see WO 88/04691), having a total volume of 48 liters,was brought to 1M (NH₄)₂ SO₄, pH 7.0. Human IL-3 was adsorbed on aFractogel TSK-butyl 650C column (d×h 25×8 cm) equilibrated with 1M(NH₄)₂ SO₄ in Tris, pH 7.0 (q 8 1/h). The column was extensively washedwith the same buffer. hIL-3 was eluted with a linear salt gradient from1.0-0.1M (NH₄)₂ SO₄ in 10 mM Tris, pH 7.0 (see FIG. 26). The hIL-3enriched fractions were further concentrated by (NH₄)₂ SO₄ precipitationat 60% saturation and the precipitate was desalted by dialysis(Spektra/Por, cutoff 3.500D) against Milli-Q water up to a conductivityof 0.70 mS or less.

Step Two: Anion Exchange Chromatography (First Run)

The hIL-3 enriched solution was brought to pH 7.8 using solid Tris andto a final conductivity of less than 0.70 mS. Subsequently it was run ona Q-Sepharose Fast Flow column (d×h 10×11 cm, q 3 1/h), equilibrated ina Tris buffer, pH 7.8, with a conductivity of less than 0.70 mS. hIL-3was collected in the run-through fraction. Most of the contaminatingprotein was bound to the column (see FIG. 27). hIL-3 was concentrated to25-30 mg/ml by ultrafiltration (Amicon, ym5 filter).

Step Three: Anion Exchange Chromatography (Second Run)

Sixty milliliters (containing 1.5-1.8 g hIL-3) of the solution obtainedfrom the previous step was run on a Q-Sepharose Fast Flow column (d×h5×90 cm, q 0.3 l/h). Several peaks with hIL-3 activity were detected inthe run-through fractions (see FIG. 28). Thus, the hIL-3 proteinfraction of interest was separated from hIL-3 degradation products. ThehIL-3 was concentrated as described above.

Step Four: Gel Filtration Chromatography

Approximately 0.9 g hIL-3 (obtained from the above steps) was run on aSephacryl S100 HR column (d×h 5×90 cm, q 0.15 l/h), equilibrated in 10mM NaH₂ PO₄, 140 mM NaCl, pH 7.0. The hIL-3 was collected in a singlepeak which was separated from a small amount of hIL-3 aggregates elutingjust before the hIl-3 (see FIG. 29).

hIL-3 obtained by this purification procedure was free from detectableamounts of contaminating protein and nucleic acid. The solutioncontaining hIL-3 was desalted and subsequently concentrated byultrafiltration to the appropriate concentration.

C. Purification of hIL-3 from E. coli (pGB/IL-302)

hIL-3 was purified from E. coli as follows. For the isolation of alacZ/hIL-3 fusion product that was intracellularly expressed in E. coli(pGB/IL-302) and stored in inclusion bodies, the cells were lysed in aTris buffer, pH 8.0, containing 1% (v/v) octanol, 1% (w/v) Tx100, 0.1%(w/v) EDTA, and 0.025% (w/v) lysozyme. 1 mM PMSF was used as aproteinase inhibitor. After an extra incubation with DNAse and Mg²⁺, thecrude inclusion bodies were centrifuged (10 min., 4000 g) and washed inthe same Tris buffer as above. After an extra centrifugation procedure(30 min., 10,000 g), inclusion body-derived protein was solubilized in 8M urea in Tris buffer under reducing conditions (5 mM dithiothreitol,DTT). Finally, centrifugation (30 min., 27,000 g) resulted in asupernatant with most of the hIL-3 that was originally present in theinclusion bodies. This solution of hIL-3 was adjusted to pH 5.0 andloaded on a Fractogel TSK CM 650M column, equilibrated in a buffercontaining 8M urea, 5 mM DTT, pH 5.0. IL-3 was eluted from this columnby a linear salt gradient from 0 to 300 mM NaCl in the same buffer. IL-3from E. coli, renaturated and solubilized in urea and partly purified byion-exchange chromatography, is positive in an AML assay.

D. Purification of hIL-3 from K. lactis (pGB/IL-316) and S. cerevisiae(pGB/IL-316)

hIL-3 was purified from yeast as follows. After centrifugation of aculture of K. lactis (pGB/IL-316), the hIL-3-containing supernatant wasfiltrated for residual cell removal, brought to a concentration of 1M(NH₄)₂ SO₄, and adjusted to pH 7.0. (1 mM PMSF was used as a proteinaseinhibitor.) This solution was loaded on a column of octyl-Sepharoseequilibrated in 1M (NH₄)₂ SO₄ in phosphate buffer, pH 7.0. Whereas mostof the protein was found in the run-through fractions, hIL-3 wasabandoned to the column. After a washing procedure with the same buffer,hIL-3 was partly released from the column in a gradient from 1M to 0M(NH₄)₂ SO₄ in phosphate buffer, pH 7.0.

The major part of hIL-3 that was still adsorbed to the column was elutedby the application of a gradient from 0% (v/v) to 100% (v/v) ethyleneglycol. The technique of hydrophobic interaction of hIL-3 onoctyl-Sepharose (or Fractogel TSK-butyl) proved to be a highly efficientfirst isolation procedure. This technique, followed by ion-exchangechromatography and gel filtration, resulted in a homogeneous preparationof hIL-3 from K. lactis. In a similar procedure using the medium from S.cerevisiae (pGB/IL-316), hIL-3 was also eluted from octyl-Sepharose in agradient from 0% (v/v) to 100% (v/v) ethylene glycol.

In an alternative procedure, hIL-3 was isolated from the cell-freemedium of a culture of K. lactis by first performing (NH₄)₂ SO₄precipitation (at 75% saturation). The collected precipitate wasresuspended in, and dialyzed against, phosphate buffer. This methodresulted in precipitation of most hIL-3 included in just a minor part ofthe total amount of protein that was originally present. ThehIL-3-containing solution was adjusted to pH 5.0 and loaded on aFractogel TSK-CM 650M cation-exchange column, equilibrated in the samebuffer. Whereas all hIL-3 was adsorbed to the column, most of thecontaminating protein was found in the run-through fractions. hIL-3 waseluted from the column by the application of a gradient from 0 mM to 300mM NaCl in the same buffer. Fractionated hIL-3 obtained by this two-stepprocedure is close to homogeneity. As a result of the nature ofglycosylation, inherent when using K. lactis as the expression system,hIL-3 purified to homogeneity was composed of different forms of theprotein with respect to molecular weight. All hIL-3 preparationsobtained from the above-mentioned yeast expression systems are positivein the AML assay.

hIL-3 from K. lactis obtained after purification on Fractogel TSK-CM650M as described above, surprisingly showed a low but distinct bindingaffinity for heparin-Sepharose as was detected by SDS-PAGE andimmunoblotting. The affinity of hIL-3 for heparin may suggest a bindingin vivo to similar matrices (the glycosaminoglycans, for instance) thatare present within the human bone marrow stroma. Binding of hIL-3 tosuch a matrix may be part of its physiological function. The presence ofheparin-like and/or glycosaminoglycan-like binding site(s) on growthfactors or other proteins could be an important mechanism for the invivo targeting of such proteins to specific matrices within the bonemarrow stroma or elsewhere in the body where their action is required.The development of second generation proteins with new or better bindingsites for the above-mentioned matrices could become a potent tool in theformulating and targeting of pharmaceutical proteins.

E. Purification of hIL-3 from Mammalian Cell Cultures

hIL-3 obtained from a mammalian cell culture was purified from themedium by using similar methods as described for the purification ofhIL-3 from B. licheniformis. Medium from transformed C127 cellscontaining pGB/IL-328 was brought to 1M (NH₄)₂ SO₄, adjusted to pH 7.0with NaOH, and loaded on a Fractogel TSK-Butyl 650C column (1 mM PMSFwas used as a proteinase inhibitor). hIL-3-enriched fractions (withabout 10% of total protein that was loaded on the column) were eluted ina gradient from 1M to 0M (NH₄)₂ SO₄ in 10 mM Tris-HCl, pH 7.0. Thesefractions were concentrated by (NH₄)₂ SO₄ precipitation at 60%saturation. The protein precipitate was dissolved and dialyzed againstH₂ O, after which 20 mM Tris-HCl, pH 7.8, was added up to a conductivityof 1.4 mS or less. The hIL-3 preparation was subsequently loaded on aFractogel TSK-DEAE 650M column. hIL-3 was partly found in therun-through fractions and was also eluted in a gradient from 0 to 200 mMNaCl. Most of the contaminating proteins bound to the column and elutedafter the hIL-3 fractions.

Again this two-step procedure, combined with (NH₄)₂ SO₄ precipitation,resulted in a highly concentrated and purified preparation of hIL-3.

EXAMPLE 10 In vivo Effect of hIL-3 in Chimpanzees

The recombinant hIL-3 produced by B. licheniformis cells was purifiedaccording to the procedure described in Example 9A. The IL-3 solutionwas then diluted in pyrogen free water to give the required finalconcentration. Chimpanzees were injected subcutaneously with thissolution. Administration of 30 ug of IL-3 per kg per day for a period ofseven days resulted in an increase of thrombocytes and a tendency toleucocytosis see FIG. 25). This surprising result of IL-3 activity as asingle agent was unanticipated and may indicate a clinical applicationfor IL-3 in thrombocytopenia.

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We claim:
 1. A method of substantially purifying a protein having humanIL-3 activity from a crude protein product obtained from a recombinantcell expressing a DNA sequence encoding a protein having the biologicalactivity of human IL-3 said method comprising:(a) performing hydrophobicinteraction chromatography on said protein product and collectingfractions comprising a protein having human IL-3 activity; followed by(b) performing ion exchange chromatography on the fractions collected instep (a) and collecting fractions comprising a protein having human IL-3activity; followed by (c) performing gel filtration chromatography onthe fractions collected in step (b) to yield a substantially purepotential having human IL-3 activity.
 2. The method of claim 1 whereinsaid recombinant cell is a bacterial cell and said crude protein productis obtained from a lysate of said recombinant cell.
 3. The method ofclaim 2 wherein said recombinant bacterial cell is E. coli transformedwith pGB/IL-302.
 4. The method of claim 1 wherein said crude proteinproduct is obtained from substantially cell-free medium in which saidrecombinant cells have been cultured.
 5. The method of claim 4 whereinsaid recombinant cell is B. licheniformis T9 transformed withpGB/IL-322.
 6. The method of claim 4 wherein said recombinant cell is ayeast cell selected from the group consisting of K. lactis transformedwith pGB/IL-316 and S. cerevisiae transformed with pGB/IL-316.
 7. Themethod of claim 4 wherein said recombinant cell is a C127 celltransformed with pGB/IL-328.
 8. A method for substantially purifying aprotein having human IL-3 activity from a crude protein product obtainedfrom a recombinant cell expressing a DNA sequence encoding a proteinhaving the biological activity of human IL-3 said method comprising:(a)precipitating said protein using (NH₄)₂ SO₄ ; followed by (b)resuspending said protein-containing precipitate and performinghydrophobic interaction chromatography on resuspended protein andcollecting fractions comprising protein having human IL-3 activity;followed by (c) performing ion exchange chromatography on the fractioncollected in step (b) and collecting fractions comprising a proteinhaving human IL-3 activity; followed by (d) performing gel filtrationchromatography on the fractions collected in step (c) to yield asubstantially purified protein having human IL-3 activity.
 9. The methodof claim 8 wherein said crude protein product is obtained fromsubstantially cell free medium in which said recombinant cells have beencultured.
 10. The method of claim 9 wherein said recombinant cell is K.lactis transformed with pGB/IL-316.
 11. A method for substantiallypurifying a protein having human IL-3 activity from a crude proteinproduct obtained from a recombinant cells expressing a DNA sequenceencoding a protein having the biological activity of human IL-3 saidmethod comprising:(a) performing hydroxylapatite chromatography on saidcrude protein product and collecting fractions comprising a proteinhaving human IL-3 activity; followed by (b) performing hydrophobicinteraction chromatography on the fractions collected in step (a) andcollecting fractions comprising protein having human IL-3 activity;followed by (c) performing ion exchange chromatography on the fractionscollected in step (b) and collecting fractions comprising protein havinghuman IL-3 activity; followed by (d) performing gel filtrationchromatography on the fractions collected in step (c) to yield asubstantially pure protein having human IL-3 activity.
 12. The method ofclaim 11 wherein said crude protein product is obtained fromsubstantially cell free medium in which said recombinant cells have beencultured.
 13. The method according to any one of claims 1, 8 or 11wherein the IL-3 protein is produced by the recombinant expression of aDNA sequence encoding the amino acid sequence shown as positions 1-133in sequence "H" of FIG.
 1. 14. The method according to any one of claims1, 8 or 11 wherein the IL-3 protein is produced by the recombinantexpression of a DNA sequence encoding the amino acid sequence shown aspotassium 1-133 in sequence "H" of FIG. 1 with the proviso that saidamino acid sequence contains a substitution selected from the groupconsisting of trp¹³ →arg¹³ ; leu⁹ →pro⁹ and trp¹³ →arg¹³ ; and met³→thr³.
 15. The method according to any one of claims 1, 8 or 11 whereinthe IL-3 protein is produced by the recombinant expression of a DNAsequence encoding the amino acid sequence shown as positions 1-133 insequence "H" of FIG. 1 wherein 1-14 amino acids at the N-terminus ofsaid sequence are substituted by other amino acids.
 16. The methodaccording to claim 15 wherein the first 14 N-terminal amino acids ofsaid sequence H are replaced by lacZ protein.
 17. The method accordingto any one of claims 1, 8 or 11 wherein the protein or fragment thereofis purified from contaminating components of the growth medium aftersecretion from the recombinant cells.
 18. The method according to claim17 wherein said purified protein or fragment thereof is a substantiallypyrogen-free product.